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Proteins overview

Proteins are essential biomolecules that make up 15% of cells and perform many critical functions. They are composed of amino acid monomers linked into polymers that fold into complex three-dimensional shapes defined by non-covalent interactions between residues. Protein structure determines its diverse functions, which include serving as enzymes, hormones, antibodies, structural components, transporters, and more. Understanding protein structure is necessary to elucidate proteins' roles at the molecular level.

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Protein Structure and Function Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India
Our life is maintained byOur life is maintained by molecular network systemsmolecular network systems Molecular network system in a cell (From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
Proteins Make up about 15% of the cell Have many functions in the cell Enzymes Structural Transport Motor Storage Signaling Receptors Gene regulation Special functions
Animals have much more proteins than in plants (Seeds) in which cellulose predominates. Among animals mammals constitutes largely proteins (Skin, hair, nails, muscles etc) Antibodies, enzymes, some harmones (insulin) are protenatious in nature Some important facts about proteins
It is very important to note htat the tissue proteins of any two of the individuals are not identical, except for two twins. Due to this characteristic, proteins help in protecting the body by the attack of foreign toxic proteins and viruses. The biological importance of proteins can be judged by the fact that the animals can live for a long time without fat or carbohydrate, but not without proteins Some important facts about proteins
Proteins mainly supply new tissue, repair working parts and make up the loss (eg as gland secretion) in the vital process. Only the plants can build up proteins from inorganic materials, like nitrates, ammonium sulphate, carbon di oxide and water While most of the animals derive them mainly from plants and some other animals Some important facts about proteins
Characteristics of proteins Most of the proteins are hydrophilic and some are hydrophobic, high polymer colloids, few such as insulin, TMV protein etc are crystalline. All proteins are leavorotatory, this property is due to the presence of alpha-amino acids. Proteins doesn’t bear any color except chromoproteins (heamoglobin and myoglobin)
They have no melting point or decomposition temperature A pure protein is tasteless and odourless. Denaturation: on heating, exposing to ultraviolate radiation or treating with number of solvents or reagents (alcohol, acetone, aqueous potassium iodide) the proteins are precipitated out and thuis undergo remarkable changes in hteir solubility, optical rotation and biological properties (eg, Enzymes become inactive when denatures. These changes may be irreversible. Characteristics of proteins
Classification of Proteins Proteins are generally classified on the basis of increasing complexity in their structures 1. Simple: which yield only alpha-amino acids on hydrolysis Albumin (soluble in water), globulin(insoluble in water), histamines etc
2. Conjugated proteins: the conjugated proteins contain simple protein along with a non protein group Glycoproteins, phosphoprteins, chromoprotein etc 3. Derived proteins: Derived proteins are the products formed by the action of physical, chemical or enzymatic agents on natural proteins Fibrous and globular proteins
Amino acid(s) mg per kg body weight mg per 70 kg mg per 100 kg Main food sources H Histidine 10 700 1000 soy protein, eggs, parmesan, sesame,  peanuts[7] I Isoleucine 20 1400 2000 eggs, soy protein & tofu, whitefish, pork , parmesan[8] L Leucine 39 2730 3900 eggs, soy protein, whitefish, parmesan , sesame[9] K Lysine 30 2100 3000 eggs, soy protein, whitefish, parmesan , smelts[10] M Methionine+ C Cysteine 10.4 + 4.1 (15 total) 1050 1500 eggs, whitefish, sesame, smelts,  soy protein[11]  + eggs, soy protein,  sesame, mustard seeds,peanuts[12] FPhenylalanine+ Y Tyrosine 25 (total) 1750 2500 eggs, soy protein, peanuts, sesame,  whitefish[13]  + soy protein, eggs,  parmesan, sesame[14] T Threonine 15 1050 1500 eggs, soy protein, whitefish, smelts,  sesame[15] W Tryptophan 4 280 400 soy protein, sesame, eggs,  winged beans, chia seeds[16] V Valine 26 1820 2600 eggs, soy protein, parmesan, sesame,  beef[17]
12 Fibrous proteins have a structural role •Collagen is the most abundant protein inCollagen is the most abundant protein in vertebrates. Collagen fibers are a majorvertebrates. Collagen fibers are a major portion of tendons, bone and skin. Alphaportion of tendons, bone and skin. Alpha helices of collagen make up a triple helixhelices of collagen make up a triple helix structure giving it tough and flexiblestructure giving it tough and flexible properties.properties. •Fibroin fibers make the silk spun by spidersFibroin fibers make the silk spun by spiders and silk worms stronger weight for weightand silk worms stronger weight for weight than steel! The soft and flexible propertiesthan steel! The soft and flexible properties come from the beta structure.come from the beta structure. •Keratin is a tough insoluble protein thatKeratin is a tough insoluble protein that makes up the quills of echidna, your hair andmakes up the quills of echidna, your hair and nails and the rattle of a rattle snake. Thenails and the rattle of a rattle snake. The structure comes from alpha helices that arestructure comes from alpha helices that are cross-linked by disulfide bonds.cross-linked by disulfide bonds.
13 The globular proteins The globular proteins have a number of biologically important roles. They include: Cell motility – proteins link together to form filaments which make movement possible. Organic catalysts in biochemical reactions – enzymes Regulatory proteins – hormones, transcription factors Membrane proteins – MHC markers, protein channels, gap junctions Defense against pathogens – poisons/toxins, antibodies, complement Transport and storage – hemoglobin and myosin
14 Proteins for cell motility Myosin (red) and actin filaments (green) in coordinated muscle contraction. Actin bound to the mysoin binding site (groove in red part of myosin protein). Add energy (ATP) and myosin moves, moving actin with it. The sperm motility is activated by changes in intracellular ion concentration. The change in concentration that signals the mechanism is different among species. In marine invertebrates and sea urchins, the rise in pH to about 7.2-7.6 activates ATPase which leads to decrease in potassium, thus induces membrane hyperpolarization. As a result, sperm motility is activated. The change in cell volume which alters intracellular ion concentration can also contribute to the activation of sperm motility. In some mammals, sperm motility is activated by increase in pH, calcium ion and cAMP,
15 Eukaryote cells have a cytoskeleton made up of straight hollow cylinders called microtubules (bottom left). They help cells maintain their shape, they act like conveyer belts moving organelles around in the cytoplasm, and they participate in forming spindle fibres in cell division. Microtubules are composed of filaments of the protein, tubulin (top left) . These filaments are compressed like springs allowing microtubules to ‘stretch and contract’. 13 of these filaments attach side to side, a little like the slats in a barrel, to form a microtubule. This barrel shaped structure gives strength to the microtubule. Tubulin forms helical filaments Proteins in the Cell Cytoskeleton
16 Catalase speeds up the breakdown of hydrogen peroxide, (H2O2) a toxic by product of metabolic reactions, to the harmless substances, water and oxygen. The reaction is extremely rapid as the enzyme lowers the energy needed to kick-start the reaction (activation energy) Energy Progress of reaction Substrate Product No catalyst =No catalyst = Input of 71kJ energy requiredInput of 71kJ energy required Activation Energy With catalaseWith catalase = Input of 8 kJ energy required= Input of 8 kJ energy required Proteins speed up reactions - EnzymesProteins speed up reactions - Enzymes +2 2
17 Proteins can regulate metabolism – hormones When your body detects an increase in the sugar content of blood after a meal, the hormone insulin is released from cells in the pancreas. Insulin binds to cell membranes and this triggers the cells to absorb glucose for use or for storage as glycogen in the liver. Proteins span membranes –protein channelsProteins span membranes –protein channels Source: http://www.biology.arizona.edu/biochemistry/tutorials/chemistry/page2.html http://www.cbp.pitt.edu/bradbury/projects.htm The CFTR membrane protein is an ion channel that regulates the flow of chloride ions. Not enough of this protein gets inserted into the membranes of people suffering Cystic fibrosis. This causes secretions to become thick as they are not hydrated. The lungs and secretory ducts become blocked as a consequence.
18 Proteins Defend us against pathogens – antibodies Left: Antibodies like IgG found in humans, recognise and bind to groups of molecules or epitopes found on foreign invaders. Right: The binding site of an antigen protein (left) interacting with the epitope of a foreign antigen (green)
Protein structure Protein structure is the biomolecular structure of a protein molecule. Each protein is a polymer – specifically a polypeptide – that is a sequence formed from various L-α-amino acids (also referred to as residues). By convention, a chain under 40 residues is often identified as a  peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations, driven by a number of non-covalent interactions such as  hydrogen bonding, ionic interactions, Van der Waals forces, and  hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, and  dual polarisation interferometry to determine the structure of proteins.
Amino acid: Basic unit ofAmino acid: Basic unit of proteinprotein COO- NH3 + C R H An amino acid Different side chains, R, determin the properties of 20 amino acids. Amino group Carboxylic acid group
20 Amino acids20 Amino acids Glycine (G) Glutamic acid (E)Asparatic acid (D) Methionine (M) Threonine (T)Serine (S)Glutamine (Q) Asparagine (N)Tryptophan (W)Phenylalanine (F) Cysteine (C) Proline (P) Leucine (L)Isoleucine (I)Valine (V)Alanine (A) Histidine (H)Lysine (K) Tyrosine (Y) Arginine (R) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
Proteins are linear polymers ofProteins are linear polymers of amino acidsamino acids R1 NH3 ＋ C CO H R2 NH C CO H R3 NH C CO H R2 NH3 ＋ C CO O ー H ＋ R1 NH3 ＋ C CO O ー H ＋ H2OH2O Peptide bond Peptide bond The amino acid sequence is called as primary structure A A F NG G S T S D K A carboxylic acid condenses with an amino group with the release of a water
Amino acid sequence isAmino acid sequence is encoded by DNA base sequenceencoded by DNA base sequence in a genein a gene ・ C G C G A A T T C G C G ・ ・ G C G C T T A A G C G C ・ DNA molecule ＝ DNA base sequence
Amino acid sequence isAmino acid sequence is encoded by DNA base sequenceencoded by DNA base sequence in a genein a gene Second letter T C A G Firstletter T TTT Phe TCT Ser TAT Tyr TGT Cys T Thirdletter TTC TCC TAC TGC C TTA Leu TCA TAA Stop TGA Stop A TTG TCG TAG TGG Trp G C CTT Leu CCT Pro CAT His CGT Arg T CTC CCC CAC CGC C CTA CCA CAA Gln CGA A CTG CCG CAG CGG G A ATT Ile ACT Thr AAT Asn AGT Ser T ATC ACC AAC AGC C ATA ACA AAA Lys AGA Arg A ATG Met ACG AAG AGG G G GTT Val GCT Ala GAT Asp GGT Gly T GTC GCC GAC GGC C GTA GCA GAA Glu GGA A GTG GCG GAG GGG G
Gene is protein’s blueprint,Gene is protein’s blueprint, genome is life’s blueprintgenome is life’s blueprint Gene GenomeDNA Protein Gene Gene Gene Gene Gene Gene GeneGene GeneGene GeneGene Gene Gene Protein Protein Protein Protein Protein ProteinProtein Protein Protein Protein Protein Protein Protein Protein
Gene is protein’s blueprint,Gene is protein’s blueprint, genome is life’s blueprintgenome is life’s blueprint Genome Gene Gene Gene Gene Gene Gene GeneGene GeneGene GeneGene Gene Gene Protein Protein Protein Protein Protein ProteinProtein Protein Protein Protein Protein Protein Protein Protein Glycolysis network
3 billion base pair => 6 G letters & 1 letter => 1 byte The whole genome can be recorded in just 10 CD-ROMs! In 2003, Human genomeIn 2003, Human genome sequence was deciphered!sequence was deciphered! Genome is the complete set of genes of a living thing. In 2003, the human genome sequencing was completed. The human genome contains about 3 billion base pairs. The number of genes is estimated to be between 20,000 to 25,000. The difference between the genome of human and that of chimpanzee is only 1.23%!
Hierarchical nature of proteinHierarchical nature of protein structurestructure Primary structure (Amino acid sequence) ↓ Secondary structure （ α-helix, β-sheet ） ↓ Tertiary structure （ Three-dimensional structure formed by assembly of secondary structures ） ↓ Quaternary structure （ Structure formed by more than one polypeptide chains ）
Basic structural units of proteins:Basic structural units of proteins: Secondary structureSecondary structure α-helix β-sheet Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.
The primary structure refers to amino acid linear sequence of the polypeptide chain. The primary structure is held together by covalent bonds such as peptide bonds, which are made during the process of protein biosynthesis or translation. The two ends of the  polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N- terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. Primary structure Post-translational modifications such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene. Example: Insulin is composed of 51 amino acids in 2 chains. One chain has 31 amino acids and the other has 20 amino acids.
Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand or  beta sheets, were suggested in 1951 by Linus Pauling and coworkers.  These secondary structures are defined by patterns of hydrogen bonds  between the main-chain peptide groups. They have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the  Ramachandran plot. Both the alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential secondary structures may form a "super secondary unit" Secondary structure
33 Alpha Helix A helix can turn right or left from N to C terminus – only right-handed are observed in nature as this produces less clashes All hydrogen bonds are satisfied except at the ends = stable
34 Alpha Helix Continued There are 3.6 residues per turn A helical wheel will outline the surface properties of the helix
35 Other (Rarer) Helix Types - 310 Less favorable geometry 3 residues per turn with i+3 not i+4 Hence narrower and more elongated Usually seen at the end of an alpha helix
36 Other (Very Rare) Helix Types - Π Less favorable geometry 4 residues per turn with i+5 not i+4 Squat and constrained
37 Beta Sheets
38 Beta Sheets Continued Between adjacent polypeptide chains Phi and psi are rotated approximately 180 degrees from each other Mixed sheets are less common Viewed end on the sheet has a right handed twist that may fold back upon itself leading to a barrel shape (a beta barrel) Beta bulge is a variant; residue on one strand forms two hydrogen bonds with residue on other – causes one strand to bulge – occurs most frequently in parallel sheets
39 Other Secondary Structures – Loop or Coil Often functionally significant Different types Hairpin loops (reverse turns) – often between anti-parallel beta strands Omega loops – beginning and end close (6-16 residues) Extended loops – more than 16 residues
Tertiary structure refers to three-dimensional structure of a single protein molecule. The alpha-helices and beta-sheets are folded into a compact globule. The folding is driven by the non-specific  hydrophobic interactions (the burial of hydrophobic residues from water), but the structure is stable only when the parts of a protein domain are locked into place by specific tertiary interactions, such as  salt bridges, hydrogen bonds, and the tight packing of side chains anddisulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment. Tertiary structure
Protein are frequently described as consisting from several structural units. A structural domain is an element of the protein's overall structure that is self-stabilizing and often folds independently of the rest of the protein chain. Many domains are not unique to the protein products of one gene or one  gene family but instead appear in a variety of proteins. Domains often are named and singled out because they figure prominently in the biological function of the protein they belong to; for example, the "calcium-binding domain of calmodulin". Because they are independently stable, domains can be "swapped" by genetic engineering between one protein and another to make chimeras. The structural and sequence motifs refer to short segments of protein three-dimensional structure or amino acid sequence that were found in a large number of different proteins. The super secondary structure refers to a specific combination of  secondary structure elements, such as beta-alpha-beta units or helix-turn-helix motif. Some of them may be also referred to as structural motifs. Protein fold refers to the general protein architecture, like helix bundle,  Domains, motifs, and folds in protein structure
42 Tertiary Structure as Dictated by the Environment Proteins exist in an aqueous environment where hydrophilic residues tend to group at the surface and hydrophobic residues form the core – but the backbone of all residues is somewhat hydrophilic – therefore it is important to have this neutralized by satisfying all hydrogen bonds as is achieved in the formation of secondary structures Polar residues must be satisfied in the same way – on occasion pockets of water (discreet from the solvent) exist as an intrinsic part of the protein to satisfy this need Ion pairs (aka salt bridge) form important interactions Disulphide linkages between cysteines form the strongest (ie covalent tertiary linkages); the majority of cysteines do not form such linkages
43 Tertiary Structure as Dictated by Protein Modification To the amino acid itself eg hydroxyproline needed for collagen formation Addition of carbohydrates (intracellular localization) Addition of lipids (binding to the membrane) Association with small molecules – notably metals eg hemoglobin
44 There are Different Forms of Classification apart from Structural Biochemical Globular Membrane Fibrous myoglobin Collagen Bacteriorhodopsin
Quaternary structure is the three-dimensional structure of a multi-subunit protein and how the subunits fit together. In this context, the quaternary structure is stabilized by the same non-covalent interactions and  disulfide bonds as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. The subunits are frequently related to one another by  symmetry operations, such as a 2-fold axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin). Quaternary structure
Three-dimensional structure ofThree-dimensional structure of proteinsproteins Tertiary structure Quaternary structure
Keratin Keratin is a family of  fibrous structural proteins. Keratin is the key structural material making up the outer layer of human skin. In general Keratin is the protein that protects the epithelial cells from damage and stress that could kill the cell. It is also the key structural component of hair and nails. Keratin monomers assemble into bundles to form  intermediate filaments, which are tough and insoluble and form strong unmineralized tissues found inreptiles, birds, amphibians, and  mammals.
The average molecular weight of Keratin-7 is 54kD · In one case, the human molecular weight of Keratin 7 was 51.4 kD. The Chromosome Location of Keratin This means that it is located on the 12th human chromosome.
alpha (cysteine rich) isomer found in cytoskeleton and hair. beta (cysteine poor) isomer found mostly in birds and reptiles. It is the building block of scales, feathers and claws. It is rich in residues with small side chains: glycine, alanine and serine. alpha form can be stretched up to 120% in moist heat. beta form is rigid. Cysteine can form disulfide bridges with other cysteine residues. These cross-linkages decrease the elasticity of alpha-keratin.
Keratin-Etymology the α-keratins in the hair (including  wool), horns, nails, claws and hooves  of mammals. the harder β-keratins found in nails and in the scales and claws of reptiles, their shells (Testudines, such as  tortoise, turtle, terrapin), and in the  feathers, beaks, claws of birds and quills of porcupines. (These keratins are formed primarily in beta sheets. However, beta sheets are also found in α-keratins.) The baleen plates of filter-feeding  whales are made of keratin.
In the early 1950s Linus Pauling and R.B. Corey in proposed several structures for keratin. Observed shorter than expected amide C-N bond. They deduced that the peptide bond was planar. A planar peptide bond reduced the number of conformations of a poly-peptide chain and led to their proposal of the alpha helix and the beta sheet. alpha-helix explained the x-ray data which showed a repeat unit of 0.50 – 0.55 nm. This distance corresponds to the height of the rise per revolution of helix. alpha-helix also explained a repeat unit of 0.15 nm. This distance corresponds to the height of the rise per residue. The ratio of these two numbers give the number of amino acids per revolution: 3.6 Hydrogen bonding occurs between carbonyl oxygen and the amide hydrogen on next twist of helix.
In a coil group of 7 residues, 1st & 4th positions contain hydrophobic aa’s These nonpolar aa’s on different helical chains attract each other and make up the inside positions of the double coils These hydrophobic reactions stabilize the coil structure The outside positions are mostly polar aa’s
Fibroin Fibroin is an insoluble protein created by spiders, the larvae of Bombyx mori, other moth genera such as Antheraea, Cricula, Samia and Gonometa, and numerous other insects. Silk in its raw state consists of two main proteins,  sericin and fibroin, fibroin being the structural center of the silk, and sericin being the sticky material surrounding it.
Hemoglobin and Myoglobin Because of its red color, the red blood pigment has been of interest since antiquity. •First protein to be crystallized - 1849. •First protein to have its mass accurately measured. •First protein to be studied by ultracentrifugation. •First protein to associated with a physiological condition. •First protein to show that a point mutation can cause problems. •First proteins to have X-ray structures determined. •Theories of cooperativity and control explain hemoglobin function
The structure of myoglobin Andrew Kendrew and Max Perutz solved the structure of these molecules in 1959 to 1968. Myoglobin: 44 x 44 x 25 Å single subunit 153 amino acid residues 121 residues are in an a helix. Helices are named A, B, C, …F. The heme pocket is surrounded by E and F but not B, C, G, also H is near the heme. Amino acids are identified by the helix and position in the helix or by the absolute numbering of the residue. shared the 1962 Nobel Prize in chemistry with
Myoglobin is the primary oxygen-carrying pigment of muscle tissues. High concentrations of myoglobin in muscle cells allow organisms to hold their breaths longer. Diving mammals such as whales and seals have muscles with particularly high myoglobin abundance Myoglobin forms pigments responsible for making meat red. The color that meat takes is partly determined by the oxidation states of the iron atom in myoglobin and the oxygen species attached to it. When meat is in its raw state, the iron atom is in the +2 oxidation state, and is bound to a dioxygen molecule (O2). Meat cooked well done is brown because the iron atom is now in the +3 oxidation state, having lost an electron, and is now coordinated by a water molecule. Under some conditions, meat can also remain pink all through cooking, despite being heated to high temperatures. If meat has been exposed to nitrites, it will remain pink because the iron atom is bound to NO, nitric oxide (true of, e.g., corned beef or cured hams).
The Backbone structure of Myoglobin 58 Heme prosthetic group
Heme Prosthetic Group Heme (Fe2+ ) has affinity for O2. Hematin (Fe3+ ) cannot bind O2. Located in crevice where it is protected from oxidation. N N N N HO O Fe
Oxygen Binding to Myoglobin O2 binds to only available coordination site on iron atom. His 93 (proximal his) binds directly to iron. His 64 (distal his) stabilizes the O2 binding site. http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_44.JPG distal histidine proximal histidine
Hemoglobin Spherical 64 x 55 x 50 Å two fold rotation of symmetry α and β subunits are similar and are placed on the vertices of a tetrahedron. There is no D helix in the α chain of hemoglobin. Extensive interactions between unlike subunits α2-β2 or α1-β1 interface has 35 residues while α1-β2 and α2-β1 have 19 residue contact. Oxygenation causes a considerable structural conformational change
O2 Binding and Allosteric Properties of Hemoglobin • Hemoglobin binds and transports HHemoglobin binds and transports H++ , O, O22 andand COCO22 in an allosteric mannerin an allosteric manner • Allosteric interaction –Allosteric interaction – of, relating to, undergoing, or being aof, relating to, undergoing, or being a change in the shape and activity of a protein (as an enzyme) that resultschange in the shape and activity of a protein (as an enzyme) that results from combination with another substance at a point other than thefrom combination with another substance at a point other than the chemically active sitechemically active site • a regulatory mechanism where a smalla regulatory mechanism where a small molecule (effector) binds and alters anmolecule (effector) binds and alters an enzymes activityenzymes activity
• There are two general structural states - the deoxy or T formThere are two general structural states - the deoxy or T form and the oxy or R form.and the oxy or R form. One type of interactions shift is the polar bonds between theOne type of interactions shift is the polar bonds between the alpha 1 and the beta 2 subunits.alpha 1 and the beta 2 subunits. The two states
The T form finds the terminals in several important H bonds and salt bridges. In the T form the C terminus of each subunit are "locked" into position through several hydrogen and ionic bonds. Shifts into the R state break these and allow an increased movement throughout the molecule. Note that binding of one or more oxygen can have a dramatic affect on the other subunits that have not yet bound an O2.
Quaternary structure of deoxy- and oxyhemoglobin T-state R-state
Oxygenation rotates the α1β1 dimer in relation to α2β2 dimer about 15° The conformation of the deoxy state is called the T state The conformation of the oxy state is called the R state individual subunits have a t or r if in the deoxy or oxy state. What causes the differences in the conformation states? It is somehow associated with the binding of oxygen, but how?
The positive cooperativity of O2 binding to Hb arises from the effect of the ligand-binding state of one heme on the ligand-binding affinity of another. The Fe iron is about 0.6 Å out of the heme plane in the deoxy state. When oxygen binds it pulls the iron back into the heme plane. Since the proximal His F8 is attached to the Fe this pulls the complete F helix like a lever on a fulcrum.
Binding of the oxygen on one heme is more difficult but its binding causes a shift in the α1-β2 contacts and moves the distal His E7 and Val E11 out of the oxygen’s path to the Fe on the other subunit. This process increases the affinity of the heme toward oxygen. The α1-β2 contacts have two stable positions. These contacts, which are joined by different but equivalent sets of hydrogen bonds and act as a binary switch between the T and the R states
The energy in the formation of the Fe-O2 bond formation drives the T→ R transition. Hemoglobins O2 -binding Cooperativity derives from the T → R Conformational shift. •The Fe of any subunit cannot move into its heme plane without the reorientation of its proximal His so as to prevent this residue from bumping into the porphyrin ring. •The proximal His is so tightly packed by its surrounding groups that it can not reorient unless this movement is accompanied by the previously described translation of the F helix across the heme plane. •The F helix translation is only possible in concert with the quaternary shift that steps the α1C-β2FG contact one turn along the α1C helix.
•The inflexibility of the α1-β1 and the α2-β2 interfaces requires that this shift simultaneously occur at both the α1-β2 and α2-β1 interfaces. No one subunit or dimer can change its conformation. The t state with reduced oxygen affinity will be changed to the r state without binding oxygen because the other subunits switch upon oxygen binding. Unbound r state has a much higher affinity for oxygen, and this is the rational for cooperativity
Hemoglobin function α2,β2 dimer which are structurally similar to myoglobin •Transports oxygen from lungs to tissues. •O2 diffusion alone is too poor for transport in larger animals. •Solubility of O2 is low in plasma i.e. 10-4 M. •But bound to hemoglobin, [O2] = 0.01 M or that of air •Two alternative O2 transporters are; •Hemocyanin, a Cu containing protein. •Hemoerythrin , a non-heme containing protein.
Function of the globin Protoporphyrin binds oxygen to the sixth ligand of Fe(II) out of the plane of the heme. The fifth ligand is a Histidine, F8 on the side across the heme plane. His F8 binds to the proximal side and the oxygen binds to the distal side. The heme alone interacts with oxygen such that the Fe(II) becomes oxidized to Fe(III) and no longer binds oxygen.
Fe O O Fe A heme dimer is formed which leads to the formation of Fe(III) By introducing steric hindrance on one side of the heme plane interaction can be prevented and oxygen binding can occur. The globin acts to: •a. Modulate oxygen binding affinity •b. Make reversible oxygen binding possible
The globin surrounds the heme like a hamburger is surrounded by a bun. Only the propionic acid side chains are exposed to the solvent. Amino acid mutations in the heme pocket can cause autooxidation of hemoglobin to form methemoglobin.
The Bohr Effect Higher pH i.e. lower [H+ ] promotes tighter binding of oxygen to hemoglobin and Lower pH i.e. higher [H+] permits the easier release of oxygen from hemoglobin ( ) ( ) + + +⇔+ xHOHbOHOHb 1n22xn2 Where n = 0, 1, 2, 3 and x ≅ 0.6 A shift in the equilibrium will influence the amount of oxygen binding. Bohr protons
Molecular chaperons
Macromolecular crowdingMacromolecular crowding When doing experiments in vitro, we should all be thinking about this: proteins in isolated (pure) systems may not behave as they do in the cell - binding partner(s) might be missing - cell conditions (pH, salts, etc) - post-translational modifications might be missing may be dramatically different This condition is known as molecular crowding
Effects of crowdingEffects of crowding Definition: Molecular crowding is a generic term for the condition where a significant volume of a solution, or cytoplasm for example, is occupied with things other than water Fact: - association constants (ka) increase significantly - dissociation constants (kd) decrease significantly (kd=1/ka) - increased on-rates for protein-protein interactions Assumption: - non-native polypeptides will have greater tendency to associate intermolecularly, enhancing the propensity of aggregation
Problem:Problem: non-native proteinsnon-native proteins • non-native proteins expose hydrophobic residues that are normally buried within the ‘core’ of the protein • these hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues - especially under crowding conditions intramolecular misfolding X X X X intermolecular aggregation X X X X X X incorrect molecular interactions & loss of activity exposed hydrophobic residues
Solution:Solution: molecular chaperonesmolecular chaperones • in the late 1970’s, the term molecular chaperone was coined to describe the properties of nucleoplasmin: Nucleoplasmin prevents incorrect interactions between histones and DNA Dictionary definition: 1: a person (as a matron) who for propriety accompanies one or more young unmarried women in public or in mixed company 2: an older person who accompanies young people at a social gathering to ensure proper behavior; broadly : one delegated to ensure proper behavior • in the late 1980’s, the term molecular chaperone was used more broadly by John Ellis to describe the roles of various cellular proteins in protein folding and assembly
Molecular chaperones:Molecular chaperones: general conceptsgeneral concepts Requirements for a protein to be considered a chaperone: (1) interacts with and stabilizes non-native forms of protein(s) - technically also: folded forms that adopt different protein conformations (2) not part of the final assembly of protein(s) Functions of a chaperone: “classical” - assist folding and assembly more recent - modulation of conformation - transport - disaggregation of protein aggregates - unfolding of proteins assisted self-assembly (as opposed to spontaneous self-assembly) assisted disassembly prevention of assembly self-assembly refers to the folding of the polypeptide, as well as to its assembly into functional homo- or hetero-oligomeric structures
Molecular chaperones:Molecular chaperones: common functional assayscommon functional assays Type of assay Rationale Binary complex formation If chaperone has high enough affinity for an unfolded polypeptide, it will form a complex detectable by: • co-migration by SEC; • co-migration by native gel electrophoresis • co-immunoprecipitation Prevention of aggregation Binding of chaperones to non-native proteins often reduces or eliminates their tendency to aggregate. Assay may detect weaker interactions than is possible with SEC Refolding Chaperones stabilize non-native proteins; some can assist the refolding of the proteins to their native state. Usually, chaperones that assist refolding are ATP-dependent Assembly Some chaperones assist protein complex assembly Activity Some chaperones modulate the conformation/activity of proteins (Miscellaneous) A number of chaperones have specialized functions
Human chaperone proteins Chaperones are found in, for example, the endoplasmic reticulum (ER), since protein synthesis often occurs in this area. Endoplasmic reticulum In the endoplasmic reticulum (ER) there are general, lectin- and non-classical molecular chaperones helping to fold proteins. •General chaperones: GRP78/BiP, GRP94, GRP170. (Binding immunoglobulin protein (BiP) also known as 78 kDa glucose-regulated protein (GRP-78) or heat shock 70 kDa protein 5 (HSPA5) is a protein that in humans is encoded by the HSPA5 gene) •Lectin chaperones: calnexin and calreticulin •Non-classical molecular chaperones: HSP47 and ERp29 •Folding chaperones: -Protein disulfide isomerase (PDI), -Peptidyl prolyl cis-trans-isomerase (PPI), -ERp57
The secondary structures that polypeptides can adopt in proteins are governed by hydrogen bonding interactions between the electronegative carbonyl oxygen atoms and the electropositive amide hydrogen atoms in the backbone chain of the molecule. These hydrogen-bonding interactions can form the framework that stabilizes the secondary structure. Many secondary structures with reasonable hydrogen bonding networks could be proposed but we see only a few possibilities in polypeptides composed of L-amino acids (proteins). Most of the possible secondary structures are not possible due to limits on the configuration of the backbone of each amino acid residue. Understanding these limitations will help to understand the secondary structures of proteins. the regions in the set of possible amino acid configurations that are allowed and disallowed in proteins. This set of values is often graphically represented as a Ramachandran diagram.
……..End……..

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Proteins overview

  • 1.
    Protein Structure and Function VijayAvin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India
  • 2.
    Our life ismaintained byOur life is maintained by molecular network systemsmolecular network systems Molecular network system in a cell (From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
  • 3.
    Proteins Make up about15% of the cell Have many functions in the cell Enzymes Structural Transport Motor Storage Signaling Receptors Gene regulation Special functions
  • 4.
    Animals have muchmore proteins than in plants (Seeds) in which cellulose predominates. Among animals mammals constitutes largely proteins (Skin, hair, nails, muscles etc) Antibodies, enzymes, some harmones (insulin) are protenatious in nature Some important facts about proteins
  • 5.
    It is veryimportant to note htat the tissue proteins of any two of the individuals are not identical, except for two twins. Due to this characteristic, proteins help in protecting the body by the attack of foreign toxic proteins and viruses. The biological importance of proteins can be judged by the fact that the animals can live for a long time without fat or carbohydrate, but not without proteins Some important facts about proteins
  • 6.
    Proteins mainly supplynew tissue, repair working parts and make up the loss (eg as gland secretion) in the vital process. Only the plants can build up proteins from inorganic materials, like nitrates, ammonium sulphate, carbon di oxide and water While most of the animals derive them mainly from plants and some other animals Some important facts about proteins
  • 7.
    Characteristics of proteins Mostof the proteins are hydrophilic and some are hydrophobic, high polymer colloids, few such as insulin, TMV protein etc are crystalline. All proteins are leavorotatory, this property is due to the presence of alpha-amino acids. Proteins doesn’t bear any color except chromoproteins (heamoglobin and myoglobin)
  • 8.
    They have nomelting point or decomposition temperature A pure protein is tasteless and odourless. Denaturation: on heating, exposing to ultraviolate radiation or treating with number of solvents or reagents (alcohol, acetone, aqueous potassium iodide) the proteins are precipitated out and thuis undergo remarkable changes in hteir solubility, optical rotation and biological properties (eg, Enzymes become inactive when denatures. These changes may be irreversible. Characteristics of proteins
  • 9.
    Classification of Proteins Proteinsare generally classified on the basis of increasing complexity in their structures 1. Simple: which yield only alpha-amino acids on hydrolysis Albumin (soluble in water), globulin(insoluble in water), histamines etc
  • 10.
    2. Conjugated proteins:the conjugated proteins contain simple protein along with a non protein group Glycoproteins, phosphoprteins, chromoprotein etc 3. Derived proteins: Derived proteins are the products formed by the action of physical, chemical or enzymatic agents on natural proteins Fibrous and globular proteins
  • 11.
    Amino acid(s) mg perkg body weight mg per 70 kg mg per 100 kg Main food sources H Histidine 10 700 1000 soy protein, eggs, parmesan, sesame,  peanuts[7] I Isoleucine 20 1400 2000 eggs, soy protein & tofu, whitefish, pork , parmesan[8] L Leucine 39 2730 3900 eggs, soy protein, whitefish, parmesan , sesame[9] K Lysine 30 2100 3000 eggs, soy protein, whitefish, parmesan , smelts[10] M Methionine+ C Cysteine 10.4 + 4.1 (15 total) 1050 1500 eggs, whitefish, sesame, smelts,  soy protein[11]  + eggs, soy protein,  sesame, mustard seeds,peanuts[12] FPhenylalanine+ Y Tyrosine 25 (total) 1750 2500 eggs, soy protein, peanuts, sesame,  whitefish[13]  + soy protein, eggs,  parmesan, sesame[14] T Threonine 15 1050 1500 eggs, soy protein, whitefish, smelts,  sesame[15] W Tryptophan 4 280 400 soy protein, sesame, eggs,  winged beans, chia seeds[16] V Valine 26 1820 2600 eggs, soy protein, parmesan, sesame,  beef[17]
  • 12.
    12 Fibrous proteins havea structural role •Collagen is the most abundant protein inCollagen is the most abundant protein in vertebrates. Collagen fibers are a majorvertebrates. Collagen fibers are a major portion of tendons, bone and skin. Alphaportion of tendons, bone and skin. Alpha helices of collagen make up a triple helixhelices of collagen make up a triple helix structure giving it tough and flexiblestructure giving it tough and flexible properties.properties. •Fibroin fibers make the silk spun by spidersFibroin fibers make the silk spun by spiders and silk worms stronger weight for weightand silk worms stronger weight for weight than steel! The soft and flexible propertiesthan steel! The soft and flexible properties come from the beta structure.come from the beta structure. •Keratin is a tough insoluble protein thatKeratin is a tough insoluble protein that makes up the quills of echidna, your hair andmakes up the quills of echidna, your hair and nails and the rattle of a rattle snake. Thenails and the rattle of a rattle snake. The structure comes from alpha helices that arestructure comes from alpha helices that are cross-linked by disulfide bonds.cross-linked by disulfide bonds.
  • 13.
    13 The globular proteins Theglobular proteins have a number of biologically important roles. They include: Cell motility – proteins link together to form filaments which make movement possible. Organic catalysts in biochemical reactions – enzymes Regulatory proteins – hormones, transcription factors Membrane proteins – MHC markers, protein channels, gap junctions Defense against pathogens – poisons/toxins, antibodies, complement Transport and storage – hemoglobin and myosin
  • 14.
    14 Proteins for cellmotility Myosin (red) and actin filaments (green) in coordinated muscle contraction. Actin bound to the mysoin binding site (groove in red part of myosin protein). Add energy (ATP) and myosin moves, moving actin with it. The sperm motility is activated by changes in intracellular ion concentration. The change in concentration that signals the mechanism is different among species. In marine invertebrates and sea urchins, the rise in pH to about 7.2-7.6 activates ATPase which leads to decrease in potassium, thus induces membrane hyperpolarization. As a result, sperm motility is activated. The change in cell volume which alters intracellular ion concentration can also contribute to the activation of sperm motility. In some mammals, sperm motility is activated by increase in pH, calcium ion and cAMP,
  • 15.
    15 Eukaryote cells havea cytoskeleton made up of straight hollow cylinders called microtubules (bottom left). They help cells maintain their shape, they act like conveyer belts moving organelles around in the cytoplasm, and they participate in forming spindle fibres in cell division. Microtubules are composed of filaments of the protein, tubulin (top left) . These filaments are compressed like springs allowing microtubules to ‘stretch and contract’. 13 of these filaments attach side to side, a little like the slats in a barrel, to form a microtubule. This barrel shaped structure gives strength to the microtubule. Tubulin forms helical filaments Proteins in the Cell Cytoskeleton
  • 16.
    16 Catalase speeds upthe breakdown of hydrogen peroxide, (H2O2) a toxic by product of metabolic reactions, to the harmless substances, water and oxygen. The reaction is extremely rapid as the enzyme lowers the energy needed to kick-start the reaction (activation energy) Energy Progress of reaction Substrate Product No catalyst =No catalyst = Input of 71kJ energy requiredInput of 71kJ energy required Activation Energy With catalaseWith catalase = Input of 8 kJ energy required= Input of 8 kJ energy required Proteins speed up reactions - EnzymesProteins speed up reactions - Enzymes +2 2
  • 17.
    17 Proteins can regulatemetabolism – hormones When your body detects an increase in the sugar content of blood after a meal, the hormone insulin is released from cells in the pancreas. Insulin binds to cell membranes and this triggers the cells to absorb glucose for use or for storage as glycogen in the liver. Proteins span membranes –protein channelsProteins span membranes –protein channels Source: http://www.biology.arizona.edu/biochemistry/tutorials/chemistry/page2.html http://www.cbp.pitt.edu/bradbury/projects.htm The CFTR membrane protein is an ion channel that regulates the flow of chloride ions. Not enough of this protein gets inserted into the membranes of people suffering Cystic fibrosis. This causes secretions to become thick as they are not hydrated. The lungs and secretory ducts become blocked as a consequence.
  • 18.
    18 Proteins Defend usagainst pathogens – antibodies Left: Antibodies like IgG found in humans, recognise and bind to groups of molecules or epitopes found on foreign invaders. Right: The binding site of an antigen protein (left) interacting with the epitope of a foreign antigen (green)
  • 19.
    Protein structure Protein structure isthe biomolecular structure of a protein molecule. Each protein is a polymer – specifically a polypeptide – that is a sequence formed from various L-α-amino acids (also referred to as residues). By convention, a chain under 40 residues is often identified as a  peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations, driven by a number of non-covalent interactions such as  hydrogen bonding, ionic interactions, Van der Waals forces, and  hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, and  dual polarisation interferometry to determine the structure of proteins.
  • 20.
    Amino acid: Basicunit ofAmino acid: Basic unit of proteinprotein COO- NH3 + C R H An amino acid Different side chains, R, determin the properties of 20 amino acids. Amino group Carboxylic acid group
  • 21.
    20 Amino acids20Amino acids Glycine (G) Glutamic acid (E)Asparatic acid (D) Methionine (M) Threonine (T)Serine (S)Glutamine (Q) Asparagine (N)Tryptophan (W)Phenylalanine (F) Cysteine (C) Proline (P) Leucine (L)Isoleucine (I)Valine (V)Alanine (A) Histidine (H)Lysine (K) Tyrosine (Y) Arginine (R) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
  • 22.
    Proteins are linearpolymers ofProteins are linear polymers of amino acidsamino acids R1 NH3 ＋ C CO H R2 NH C CO H R3 NH C CO H R2 NH3 ＋ C CO O ー H ＋ R1 NH3 ＋ C CO O ー H ＋ H2OH2O Peptide bond Peptide bond The amino acid sequence is called as primary structure A A F NG G S T S D K A carboxylic acid condenses with an amino group with the release of a water
  • 23.
    Amino acid sequenceisAmino acid sequence is encoded by DNA base sequenceencoded by DNA base sequence in a genein a gene ・ C G C G A A T T C G C G ・ ・ G C G C T T A A G C G C ・ DNA molecule ＝ DNA base sequence
  • 24.
    Amino acid sequenceisAmino acid sequence is encoded by DNA base sequenceencoded by DNA base sequence in a genein a gene Second letter T C A G Firstletter T TTT Phe TCT Ser TAT Tyr TGT Cys T Thirdletter TTC TCC TAC TGC C TTA Leu TCA TAA Stop TGA Stop A TTG TCG TAG TGG Trp G C CTT Leu CCT Pro CAT His CGT Arg T CTC CCC CAC CGC C CTA CCA CAA Gln CGA A CTG CCG CAG CGG G A ATT Ile ACT Thr AAT Asn AGT Ser T ATC ACC AAC AGC C ATA ACA AAA Lys AGA Arg A ATG Met ACG AAG AGG G G GTT Val GCT Ala GAT Asp GGT Gly T GTC GCC GAC GGC C GTA GCA GAA Glu GGA A GTG GCG GAG GGG G
  • 25.
    Gene is protein’sblueprint,Gene is protein’s blueprint, genome is life’s blueprintgenome is life’s blueprint Gene GenomeDNA Protein Gene Gene Gene Gene Gene Gene GeneGene GeneGene GeneGene Gene Gene Protein Protein Protein Protein Protein ProteinProtein Protein Protein Protein Protein Protein Protein Protein
  • 26.
    Gene is protein’sblueprint,Gene is protein’s blueprint, genome is life’s blueprintgenome is life’s blueprint Genome Gene Gene Gene Gene Gene Gene GeneGene GeneGene GeneGene Gene Gene Protein Protein Protein Protein Protein ProteinProtein Protein Protein Protein Protein Protein Protein Protein Glycolysis network
  • 27.
    3 billion basepair => 6 G letters & 1 letter => 1 byte The whole genome can be recorded in just 10 CD-ROMs! In 2003, Human genomeIn 2003, Human genome sequence was deciphered!sequence was deciphered! Genome is the complete set of genes of a living thing. In 2003, the human genome sequencing was completed. The human genome contains about 3 billion base pairs. The number of genes is estimated to be between 20,000 to 25,000. The difference between the genome of human and that of chimpanzee is only 1.23%!
  • 28.
    Hierarchical nature ofproteinHierarchical nature of protein structurestructure Primary structure (Amino acid sequence) ↓ Secondary structure （ α-helix, β-sheet ） ↓ Tertiary structure （ Three-dimensional structure formed by assembly of secondary structures ） ↓ Quaternary structure （ Structure formed by more than one polypeptide chains ）
  • 29.
    Basic structural unitsof proteins:Basic structural units of proteins: Secondary structureSecondary structure α-helix β-sheet Secondary structures, α-helix and β-sheet, have regular hydrogen-bonding patterns.
  • 30.
    The primary structure refers toamino acid linear sequence of the polypeptide chain. The primary structure is held together by covalent bonds such as peptide bonds, which are made during the process of protein biosynthesis or translation. The two ends of the  polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N- terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. Primary structure Post-translational modifications such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene. Example: Insulin is composed of 51 amino acids in 2 chains. One chain has 31 amino acids and the other has 20 amino acids.
  • 32.
    Secondary structure refers tohighly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand or  beta sheets, were suggested in 1951 by Linus Pauling and coworkers.  These secondary structures are defined by patterns of hydrogen bonds  between the main-chain peptide groups. They have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the  Ramachandran plot. Both the alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential secondary structures may form a "super secondary unit" Secondary structure
  • 33.
    33 Alpha Helix A helixcan turn right or left from N to C terminus – only right-handed are observed in nature as this produces less clashes All hydrogen bonds are satisfied except at the ends = stable
  • 34.
    34 Alpha Helix Continued Thereare 3.6 residues per turn A helical wheel will outline the surface properties of the helix
  • 35.
    35 Other (Rarer) HelixTypes - 310 Less favorable geometry 3 residues per turn with i+3 not i+4 Hence narrower and more elongated Usually seen at the end of an alpha helix
  • 36.
    36 Other (Very Rare)Helix Types - Π Less favorable geometry 4 residues per turn with i+5 not i+4 Squat and constrained
  • 37.
  • 38.
    38 Beta Sheets Continued Betweenadjacent polypeptide chains Phi and psi are rotated approximately 180 degrees from each other Mixed sheets are less common Viewed end on the sheet has a right handed twist that may fold back upon itself leading to a barrel shape (a beta barrel) Beta bulge is a variant; residue on one strand forms two hydrogen bonds with residue on other – causes one strand to bulge – occurs most frequently in parallel sheets
  • 39.
    39 Other Secondary Structures– Loop or Coil Often functionally significant Different types Hairpin loops (reverse turns) – often between anti-parallel beta strands Omega loops – beginning and end close (6-16 residues) Extended loops – more than 16 residues
  • 40.
    Tertiary structure refers tothree-dimensional structure of a single protein molecule. The alpha-helices and beta-sheets are folded into a compact globule. The folding is driven by the non-specific  hydrophobic interactions (the burial of hydrophobic residues from water), but the structure is stable only when the parts of a protein domain are locked into place by specific tertiary interactions, such as  salt bridges, hydrogen bonds, and the tight packing of side chains anddisulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment. Tertiary structure
  • 41.
    Protein are frequentlydescribed as consisting from several structural units. A structural domain is an element of the protein's overall structure that is self-stabilizing and often folds independently of the rest of the protein chain. Many domains are not unique to the protein products of one gene or one  gene family but instead appear in a variety of proteins. Domains often are named and singled out because they figure prominently in the biological function of the protein they belong to; for example, the "calcium-binding domain of calmodulin". Because they are independently stable, domains can be "swapped" by genetic engineering between one protein and another to make chimeras. The structural and sequence motifs refer to short segments of protein three-dimensional structure or amino acid sequence that were found in a large number of different proteins. The super secondary structure refers to a specific combination of  secondary structure elements, such as beta-alpha-beta units or helix-turn-helix motif. Some of them may be also referred to as structural motifs. Protein fold refers to the general protein architecture, like helix bundle,  Domains, motifs, and folds in protein structure
  • 42.
    42 Tertiary Structure asDictated by the Environment Proteins exist in an aqueous environment where hydrophilic residues tend to group at the surface and hydrophobic residues form the core – but the backbone of all residues is somewhat hydrophilic – therefore it is important to have this neutralized by satisfying all hydrogen bonds as is achieved in the formation of secondary structures Polar residues must be satisfied in the same way – on occasion pockets of water (discreet from the solvent) exist as an intrinsic part of the protein to satisfy this need Ion pairs (aka salt bridge) form important interactions Disulphide linkages between cysteines form the strongest (ie covalent tertiary linkages); the majority of cysteines do not form such linkages
  • 43.
    43 Tertiary Structure asDictated by Protein Modification To the amino acid itself eg hydroxyproline needed for collagen formation Addition of carbohydrates (intracellular localization) Addition of lipids (binding to the membrane) Association with small molecules – notably metals eg hemoglobin
  • 44.
    44 There are DifferentForms of Classification apart from Structural Biochemical Globular Membrane Fibrous myoglobin Collagen Bacteriorhodopsin
  • 45.
    Quaternary structure isthe three-dimensional structure of a multi-subunit protein and how the subunits fit together. In this context, the quaternary structure is stabilized by the same non-covalent interactions and  disulfide bonds as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. The subunits are frequently related to one another by  symmetry operations, such as a 2-fold axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin). Quaternary structure
  • 46.
    Three-dimensional structure ofThree-dimensionalstructure of proteinsproteins Tertiary structure Quaternary structure
  • 47.
    Keratin Keratin is a familyof  fibrous structural proteins. Keratin is the key structural material making up the outer layer of human skin. In general Keratin is the protein that protects the epithelial cells from damage and stress that could kill the cell. It is also the key structural component of hair and nails. Keratin monomers assemble into bundles to form  intermediate filaments, which are tough and insoluble and form strong unmineralized tissues found inreptiles, birds, amphibians, and  mammals.
  • 48.
    The average molecularweight of Keratin-7 is 54kD · In one case, the human molecular weight of Keratin 7 was 51.4 kD. The Chromosome Location of Keratin This means that it is located on the 12th human chromosome.
  • 49.
    alpha (cysteine rich)isomer found in cytoskeleton and hair. beta (cysteine poor) isomer found mostly in birds and reptiles. It is the building block of scales, feathers and claws. It is rich in residues with small side chains: glycine, alanine and serine. alpha form can be stretched up to 120% in moist heat. beta form is rigid. Cysteine can form disulfide bridges with other cysteine residues. These cross-linkages decrease the elasticity of alpha-keratin.
  • 50.
    Keratin-Etymology the α-keratins in the hair (including  wool), horns, nails, claws and hooves  of mammals. theharder β-keratins found in nails and in the scales and claws of reptiles, their shells (Testudines, such as  tortoise, turtle, terrapin), and in the  feathers, beaks, claws of birds and quills of porcupines. (These keratins are formed primarily in beta sheets. However, beta sheets are also found in α-keratins.) The baleen plates of filter-feeding  whales are made of keratin.
  • 52.
    In the early1950s Linus Pauling and R.B. Corey in proposed several structures for keratin. Observed shorter than expected amide C-N bond. They deduced that the peptide bond was planar. A planar peptide bond reduced the number of conformations of a poly-peptide chain and led to their proposal of the alpha helix and the beta sheet. alpha-helix explained the x-ray data which showed a repeat unit of 0.50 – 0.55 nm. This distance corresponds to the height of the rise per revolution of helix. alpha-helix also explained a repeat unit of 0.15 nm. This distance corresponds to the height of the rise per residue. The ratio of these two numbers give the number of amino acids per revolution: 3.6 Hydrogen bonding occurs between carbonyl oxygen and the amide hydrogen on next twist of helix.
  • 53.
    In a coilgroup of 7 residues, 1st & 4th positions contain hydrophobic aa’s These nonpolar aa’s on different helical chains attract each other and make up the inside positions of the double coils These hydrophobic reactions stabilize the coil structure The outside positions are mostly polar aa’s
  • 54.
    Fibroin Fibroin is an insolubleprotein created by spiders, the larvae of Bombyx mori, other moth genera such as Antheraea, Cricula, Samia and Gonometa, and numerous other insects. Silk in its raw state consists of two main proteins,  sericin and fibroin, fibroin being the structural center of the silk, and sericin being the sticky material surrounding it.
  • 55.
    Hemoglobin and Myoglobin Becauseof its red color, the red blood pigment has been of interest since antiquity. •First protein to be crystallized - 1849. •First protein to have its mass accurately measured. •First protein to be studied by ultracentrifugation. •First protein to associated with a physiological condition. •First protein to show that a point mutation can cause problems. •First proteins to have X-ray structures determined. •Theories of cooperativity and control explain hemoglobin function
  • 56.
    The structure ofmyoglobin Andrew Kendrew and Max Perutz solved the structure of these molecules in 1959 to 1968. Myoglobin: 44 x 44 x 25 Å single subunit 153 amino acid residues 121 residues are in an a helix. Helices are named A, B, C, …F. The heme pocket is surrounded by E and F but not B, C, G, also H is near the heme. Amino acids are identified by the helix and position in the helix or by the absolute numbering of the residue. shared the 1962 Nobel Prize in chemistry with
  • 57.
    Myoglobin is theprimary oxygen-carrying pigment of muscle tissues. High concentrations of myoglobin in muscle cells allow organisms to hold their breaths longer. Diving mammals such as whales and seals have muscles with particularly high myoglobin abundance Myoglobin forms pigments responsible for making meat red. The color that meat takes is partly determined by the oxidation states of the iron atom in myoglobin and the oxygen species attached to it. When meat is in its raw state, the iron atom is in the +2 oxidation state, and is bound to a dioxygen molecule (O2). Meat cooked well done is brown because the iron atom is now in the +3 oxidation state, having lost an electron, and is now coordinated by a water molecule. Under some conditions, meat can also remain pink all through cooking, despite being heated to high temperatures. If meat has been exposed to nitrites, it will remain pink because the iron atom is bound to NO, nitric oxide (true of, e.g., corned beef or cured hams).
  • 58.
    The Backbone structureof Myoglobin 58 Heme prosthetic group
  • 59.
    Heme Prosthetic Group Heme(Fe2+ ) has affinity for O2. Hematin (Fe3+ ) cannot bind O2. Located in crevice where it is protected from oxidation. N N N N HO O Fe
  • 60.
    Oxygen Binding toMyoglobin O2 binds to only available coordination site on iron atom. His 93 (proximal his) binds directly to iron. His 64 (distal his) stabilizes the O2 binding site. http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_44.JPG distal histidine proximal histidine
  • 61.
    Hemoglobin Spherical 64 x55 x 50 Å two fold rotation of symmetry α and β subunits are similar and are placed on the vertices of a tetrahedron. There is no D helix in the α chain of hemoglobin. Extensive interactions between unlike subunits α2-β2 or α1-β1 interface has 35 residues while α1-β2 and α2-β1 have 19 residue contact. Oxygenation causes a considerable structural conformational change
  • 62.
    O2 Binding andAllosteric Properties of Hemoglobin • Hemoglobin binds and transports HHemoglobin binds and transports H++ , O, O22 andand COCO22 in an allosteric mannerin an allosteric manner • Allosteric interaction –Allosteric interaction – of, relating to, undergoing, or being aof, relating to, undergoing, or being a change in the shape and activity of a protein (as an enzyme) that resultschange in the shape and activity of a protein (as an enzyme) that results from combination with another substance at a point other than thefrom combination with another substance at a point other than the chemically active sitechemically active site • a regulatory mechanism where a smalla regulatory mechanism where a small molecule (effector) binds and alters anmolecule (effector) binds and alters an enzymes activityenzymes activity
  • 63.
    • There aretwo general structural states - the deoxy or T formThere are two general structural states - the deoxy or T form and the oxy or R form.and the oxy or R form. One type of interactions shift is the polar bonds between theOne type of interactions shift is the polar bonds between the alpha 1 and the beta 2 subunits.alpha 1 and the beta 2 subunits. The two states
  • 64.
    The T formfinds the terminals in several important H bonds and salt bridges. In the T form the C terminus of each subunit are "locked" into position through several hydrogen and ionic bonds. Shifts into the R state break these and allow an increased movement throughout the molecule. Note that binding of one or more oxygen can have a dramatic affect on the other subunits that have not yet bound an O2.
  • 65.
    Quaternary structure ofdeoxy- and oxyhemoglobin T-state R-state
  • 66.
    Oxygenation rotates theα1β1 dimer in relation to α2β2 dimer about 15° The conformation of the deoxy state is called the T state The conformation of the oxy state is called the R state individual subunits have a t or r if in the deoxy or oxy state. What causes the differences in the conformation states? It is somehow associated with the binding of oxygen, but how?
  • 67.
    The positive cooperativityof O2 binding to Hb arises from the effect of the ligand-binding state of one heme on the ligand-binding affinity of another. The Fe iron is about 0.6 Å out of the heme plane in the deoxy state. When oxygen binds it pulls the iron back into the heme plane. Since the proximal His F8 is attached to the Fe this pulls the complete F helix like a lever on a fulcrum.
  • 69.
    Binding of theoxygen on one heme is more difficult but its binding causes a shift in the α1-β2 contacts and moves the distal His E7 and Val E11 out of the oxygen’s path to the Fe on the other subunit. This process increases the affinity of the heme toward oxygen. The α1-β2 contacts have two stable positions. These contacts, which are joined by different but equivalent sets of hydrogen bonds and act as a binary switch between the T and the R states
  • 70.
    The energy inthe formation of the Fe-O2 bond formation drives the T→ R transition. Hemoglobins O2 -binding Cooperativity derives from the T → R Conformational shift. •The Fe of any subunit cannot move into its heme plane without the reorientation of its proximal His so as to prevent this residue from bumping into the porphyrin ring. •The proximal His is so tightly packed by its surrounding groups that it can not reorient unless this movement is accompanied by the previously described translation of the F helix across the heme plane. •The F helix translation is only possible in concert with the quaternary shift that steps the α1C-β2FG contact one turn along the α1C helix.
  • 71.
    •The inflexibility ofthe α1-β1 and the α2-β2 interfaces requires that this shift simultaneously occur at both the α1-β2 and α2-β1 interfaces. No one subunit or dimer can change its conformation. The t state with reduced oxygen affinity will be changed to the r state without binding oxygen because the other subunits switch upon oxygen binding. Unbound r state has a much higher affinity for oxygen, and this is the rational for cooperativity
  • 72.
    Hemoglobin function α2,β2 dimerwhich are structurally similar to myoglobin •Transports oxygen from lungs to tissues. •O2 diffusion alone is too poor for transport in larger animals. •Solubility of O2 is low in plasma i.e. 10-4 M. •But bound to hemoglobin, [O2] = 0.01 M or that of air •Two alternative O2 transporters are; •Hemocyanin, a Cu containing protein. •Hemoerythrin , a non-heme containing protein.
  • 73.
    Function of theglobin Protoporphyrin binds oxygen to the sixth ligand of Fe(II) out of the plane of the heme. The fifth ligand is a Histidine, F8 on the side across the heme plane. His F8 binds to the proximal side and the oxygen binds to the distal side. The heme alone interacts with oxygen such that the Fe(II) becomes oxidized to Fe(III) and no longer binds oxygen.
  • 74.
    Fe O OFe A heme dimer is formed which leads to the formation of Fe(III) By introducing steric hindrance on one side of the heme plane interaction can be prevented and oxygen binding can occur. The globin acts to: •a. Modulate oxygen binding affinity •b. Make reversible oxygen binding possible
  • 75.
    The globin surroundsthe heme like a hamburger is surrounded by a bun. Only the propionic acid side chains are exposed to the solvent. Amino acid mutations in the heme pocket can cause autooxidation of hemoglobin to form methemoglobin.
  • 76.
    The Bohr Effect HigherpH i.e. lower [H+ ] promotes tighter binding of oxygen to hemoglobin and Lower pH i.e. higher [H+] permits the easier release of oxygen from hemoglobin ( ) ( ) + + +⇔+ xHOHbOHOHb 1n22xn2 Where n = 0, 1, 2, 3 and x ≅ 0.6 A shift in the equilibrium will influence the amount of oxygen binding. Bohr protons
  • 77.
  • 78.
    Macromolecular crowdingMacromolecular crowding Whendoing experiments in vitro, we should all be thinking about this: proteins in isolated (pure) systems may not behave as they do in the cell - binding partner(s) might be missing - cell conditions (pH, salts, etc) - post-translational modifications might be missing may be dramatically different This condition is known as molecular crowding
  • 79.
    Effects of crowdingEffectsof crowding Definition: Molecular crowding is a generic term for the condition where a significant volume of a solution, or cytoplasm for example, is occupied with things other than water Fact: - association constants (ka) increase significantly - dissociation constants (kd) decrease significantly (kd=1/ka) - increased on-rates for protein-protein interactions Assumption: - non-native polypeptides will have greater tendency to associate intermolecularly, enhancing the propensity of aggregation
  • 80.
    Problem:Problem: non-native proteinsnon-nativeproteins • non-native proteins expose hydrophobic residues that are normally buried within the ‘core’ of the protein • these hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues - especially under crowding conditions intramolecular misfolding X X X X intermolecular aggregation X X X X X X incorrect molecular interactions & loss of activity exposed hydrophobic residues
  • 81.
    Solution:Solution: molecular chaperonesmolecularchaperones • in the late 1970’s, the term molecular chaperone was coined to describe the properties of nucleoplasmin: Nucleoplasmin prevents incorrect interactions between histones and DNA Dictionary definition: 1: a person (as a matron) who for propriety accompanies one or more young unmarried women in public or in mixed company 2: an older person who accompanies young people at a social gathering to ensure proper behavior; broadly : one delegated to ensure proper behavior • in the late 1980’s, the term molecular chaperone was used more broadly by John Ellis to describe the roles of various cellular proteins in protein folding and assembly
  • 82.
    Molecular chaperones:Molecular chaperones: generalconceptsgeneral concepts Requirements for a protein to be considered a chaperone: (1) interacts with and stabilizes non-native forms of protein(s) - technically also: folded forms that adopt different protein conformations (2) not part of the final assembly of protein(s) Functions of a chaperone: “classical” - assist folding and assembly more recent - modulation of conformation - transport - disaggregation of protein aggregates - unfolding of proteins assisted self-assembly (as opposed to spontaneous self-assembly) assisted disassembly prevention of assembly self-assembly refers to the folding of the polypeptide, as well as to its assembly into functional homo- or hetero-oligomeric structures
  • 83.
    Molecular chaperones:Molecular chaperones: commonfunctional assayscommon functional assays Type of assay Rationale Binary complex formation If chaperone has high enough affinity for an unfolded polypeptide, it will form a complex detectable by: • co-migration by SEC; • co-migration by native gel electrophoresis • co-immunoprecipitation Prevention of aggregation Binding of chaperones to non-native proteins often reduces or eliminates their tendency to aggregate. Assay may detect weaker interactions than is possible with SEC Refolding Chaperones stabilize non-native proteins; some can assist the refolding of the proteins to their native state. Usually, chaperones that assist refolding are ATP-dependent Assembly Some chaperones assist protein complex assembly Activity Some chaperones modulate the conformation/activity of proteins (Miscellaneous) A number of chaperones have specialized functions
  • 84.
    Human chaperone proteins Chaperonesare found in, for example, the endoplasmic reticulum (ER), since protein synthesis often occurs in this area. Endoplasmic reticulum In the endoplasmic reticulum (ER) there are general, lectin- and non-classical molecular chaperones helping to fold proteins. •General chaperones: GRP78/BiP, GRP94, GRP170. (Binding immunoglobulin protein (BiP) also known as 78 kDa glucose-regulated protein (GRP-78) or heat shock 70 kDa protein 5 (HSPA5) is a protein that in humans is encoded by the HSPA5 gene) •Lectin chaperones: calnexin and calreticulin •Non-classical molecular chaperones: HSP47 and ERp29 •Folding chaperones: -Protein disulfide isomerase (PDI), -Peptidyl prolyl cis-trans-isomerase (PPI), -ERp57
  • 85.
    The secondary structuresthat polypeptides can adopt in proteins are governed by hydrogen bonding interactions between the electronegative carbonyl oxygen atoms and the electropositive amide hydrogen atoms in the backbone chain of the molecule. These hydrogen-bonding interactions can form the framework that stabilizes the secondary structure. Many secondary structures with reasonable hydrogen bonding networks could be proposed but we see only a few possibilities in polypeptides composed of L-amino acids (proteins). Most of the possible secondary structures are not possible due to limits on the configuration of the backbone of each amino acid residue. Understanding these limitations will help to understand the secondary structures of proteins. the regions in the set of possible amino acid configurations that are allowed and disallowed in proteins. This set of values is often graphically represented as a Ramachandran diagram.
  • 86.

Editor's Notes

  • #83 self-assembly refers to the folding of the polypeptide, as well as to its assembly into homo- or hetero-oligomeric structures