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Protein structure

This document outlines the goals and key concepts regarding protein structure. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Methods for determining protein structure are also covered, including protein purification techniques like chromatography, electrophoresis, and centrifugation. Protein sequencing methods such as Edman degradation are also summarized. The document provides an overview of protein structure and analysis.

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Protein Structure Student Edition 5/23/13 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall chazotte@campbell.edu Web Site: http://www.campbell.edu/faculty/chazotte Original material only ©2004-14 B. Chazotte
Goals •Understand the bases of & differences between primary, secondary, tertiary, & quaternary protein structure. •Be familiar with basic protein purification/sequencing methods & how they depend on the physical & chemical properties of proteins. •Understand the physical and chemical forces that determine secondary structure, including the peptide bond. Learn the basic types of secondary structure: -helix, -sheet, random coil & non-repetitive structures. •Understand the physical & chemical forces that determine tertiary structure - protein domains and motifs. •Be acquainted with techniques like NMR & X-ray crystallography that help determine protein structure. •Understand the physical and chemical forces that determine quaternary structure – protein folding, denaturation, renaturation, hydropathy plots. •Remember how protein structure and structural changes reflect the influence of thermodynamic concepts on structural stability.
Levels of Protein Structure •Primary •Secondary •Tertiary •Quaternary Refer to the three- dimensional shape of folded polypeptide chains
Protein Diversity For a protein of n residues there are 20n possible sequences For 40 residue protein 1.1 x 1052 For 100 residue protein 1.27 x 10130 Definitions •Peptides – typically less than 40 residues Dipeptide: 2 amino acids; Tripeptide: 3 amino acids Oligopeptide: many amino acids •Proteins – typically polypeptides with 40 or more residues •Multisubunit proteins - proteins with several identical or nonidentical subunits.
PRIMARY STRUCTURE & ANALYSIS Primary Structure: the amino acid sequence of a protein’s polypeptide chain or chains. Sometimes referred to as the covalent structure.
Conjecture on the Limitations of Protein Size Minimum: 40 residues – near the limit for a polypeptide to be able to fold into a discrete stable shape that permits it to carry out its basic function. Maximum: ~1000 residues – near the limit for the protein synthetic machinery to produce a peptide with minimal errors in the polypeptide, mRNA template, or gene DNA.
Logic of Amino Acid Sequences in Proteins The characteristics of a protein depend more on the sequence of amino acids rather than its composition. The presence of an amino acid with its characteristic physical & chemical properties at a particular place in a protein influences the protein’s properties. (review Amino Acids lecture) The 3-D shape of a protein is a consequence of the intermolecular forces among its various residues. (review Chemical Bonding lecture) Voet, Voet & Pratt 2013 Chap. 5.1
Primary Structure of Bovine Insulin Voet, Voet & Pratt 2013 Figure 5.1
Studying Proteins by Isolating Them
Protein Purification Crude Extract – 1st step whether protein is from tissue or microbe, break open the cell and release the proteins into solution. Fractionation – step where proteins are separated into different fractions bases on some chemical or physical property such as size or charge. May utilize protein solubility i.e. (pH), salt concentration, temperature, etc.
Proteins Must be Stabilized after Isolation Care must be taken to preserve protein structure and function after its is removed from its natural environment were it was stable. •pH To prevent denaturation (loss of structure) or function proteins are placed in buffered solutions at or near their native pH. •Temperature Protein purification is normally carried out at low temperature ~0º C. While some proteins are thermally stable at high temperatures, others may be affected by temperature a few degrees higher than the native environment. •Degradative Enzymes During isolation various nucleases and proteases are released from their places in the cell and can degrade nucleic acids or proteins unless temperature, pH or inhibitory agents are added. •Adsorption to Surfaces Solutions are handled to minimize foaming and are kept concentrated as interfaces (air-water, glass, plastic) can cause denaturation. •Storage To maintain protein stability. Cold (-70º C or -196º C liq N2), sometime under N2(g) to remove oxygen and prevent slow oxidation. Some of the goals are to minimize microbial growth and/or oxidation. Voet, Voet & Pratt 2013 p.96
Assay of Purified Proteins To purify a protein it is necessary to measure how much you have  need a specific assay. •Easier for enzymes as they produce a product proportional to the amount of enzyme present. •Colored or fluorescent products are especially helpful •Can also use a coupled enzyme reaction, i.e. 2nd enzyme •Can use immunochemical assays.
ELISA Enzyme-Linked Immunoabsorbent Assay Voet, Voet & Pratt 2013 Figure 5.3
Some Separation Techniques Charge Ion Exchange Chromatography Electrophoresis Polarity Hydrophobic Interaction Chromatography Size Gel Filtration Chromatography SDS-Polyacrylamide Electrophoresis Ultracentrifugation Binding Specificity Affinity Chromatography These separation techniques utilize differences in the physical and/or chemical properties that arise from the differences amino acid composition.
Protein Fractionation by Salting Out Voet, Voet & Pratt 2013 Figure 5.5 Protein solubility depends on: •Concentration of dissolved salts •Solvent polarity •pH •Temperature By careful manipulation of these properties it is possible to selectively precipitate out certain proteins and leave the other soluble.
Protein Separation by Ion Exchange Chromatography Voet, Voet & Pratt 2013 Figure 5.6 Ion exchange chromatography makes use of the fact that opposite charges attract Polyelectrolytes such as proteins that have both negative and positive charges will bind to cation or anion exchangers depending on the protein’s net charge The binding affinity (Strength of binding) depends on the presence of other ions that compete with the protein for binding sites on the immobile phase and the pH which in terms effects the protein’s net charge. Anion exchanger: e.g., DEAE Matrix–CH2-CH2-NH(CH2CH3)2 + Cation exchanger: e.g., CM Matrix-CH2COO-
Protein Separation by Gel Filtration Chromatography Voet, Voet & Pratt 2013 Figure 5.7 A bead can have different pore sizes (holes) depending on the extent of cross-linking in its component polymer. The larger proteins that are excluded from the beads have a shorter path and leave the column sooner.
Protein Separation by Affinity Chromatography Voet, Voet & Pratt 2013 Figure 5.8 Utilize the ability of certain proteins (via biochemical properties) that are able to bind specific molecules non-covalently. Bind a specific molecule called a ligand to an inert matrix – immobile phase Column conditions are then changed, e.g. pH, ionic strength or high ligand concentration, to permit the protein to elute in a highly purified form.
SDS-PAGE of Supernatants & Membrane Fraction from a Bacterium Voet, Voet & Pratt 2006 Figure 5.9 & 2013 Fig 5.9 & 5.10 Gel electrophoresis - a molecular sieving approach SDS, sodium dodecyl sulfate, when added to a protein solution binds 1 molecule of SDS per two amino acids or 1.4g per g protein.
Separation by Zonal Centrifugation Voet, Voet & Pratt 2013 Figure 5.12 Gradient preparation Berg, Tymoczko, & Stryer 2012 Fig 3.16
Protein Sequencing 1. Sequence is a prerequisite for determining protein’s 3-D structure and understanding its molecular mechanism. 2. Sequence comparisons among analogous proteins from different species yield insights into protein function as well as reveal evolutionary relationships among proteins 3. Many inherited diseases are caused by point mutations in the amino acid sequence. Sequence analysis can assist with diagnostic testing and therapy development. Voet, Voet & Pratt 2006 Chapter 5 A protein must be broken down into fragments small enough to be individually sequenced. The fragments are used to reconstruct the protein by analyzing the fragment overlaps.
Sanger Method for Protein Sequencing Voet, Voet & Pratt 2013 p.108 Box 5.1 Bind to terminal amino groups to form a yellow dinitrophenyl derivative Hydrolyze protein Identify terminal amino acid chromatographically Also today nucleic acids sequencing is frequently used to determine protein sequences
Basic Logic of Protein Sequencing Voet, Voet & Pratt 2013 Figure 5.13 1. polypeptide chains that are linked by disulfide bonds are separated by reduction of the sulfhydral groups of cysteine. 2. chemical or enzymatic means are used to cleave the resultant polypeptide chains into smaller fragments. 3. Each small fragment is sequenced. 4. Compare overlapping sequences produced by different enzymes or chemical degradations to logically reconstruct original protein sequence 5. Repeat process without cleaving the disulfide bonds to determine where those bonds are located
End Group Analysis Used to determine the number of distinct polypeptide chains in a protein (if end groups not chemically blocked). •There are several procedures for the N-terminus. •No reliable chemical procedure for C-terminus, an enzymatic approach uses carboxypeptidases.
Protein Sequencing End Group Analysis with Danzyl Chloride Voet, Voet & Pratt 2013 Figure 5.14 Conjugate the fluorophore to primary amine(s). Perform an acid hydrolysis Identify via chromatography the labeled amino acid.
Disulfide Bond Cleavage Need to cleave to separate polypeptide chains. Two methods: • oxidative cleavage with performic acid disadvantage: destroys met and Trp indol side chain • reductive cleavage with mercaptan e.g. 2-mercaptoethanol. Usually alkylate product with iodoacetic acid to prevent disulfide bond reformation.
Peptide Hydrolysis Chemical approach Acid hydrolysis disadvantages – destroys Ser, Thr, Tyr & Trp converts Asn & Gln to Asp & Glu, respectively Base Hydrolysis disadvantages – destroys Cys, Ser, Thr & Arg Biochemical Approach Enzymatic Hydrolysis disadvantages – often incomplete some autodigestion
Amino Acid Analysis Separation by HPLC Voet, Voet & Pratt 2006 Figure 5.15 Complete hydrolysis will yield the composition but not the sequence
Molecular Mass Determination by Mass Spectrometry Voet, Voet, & Pratt 2013 Fig 17a,bBerg, Tymoczko, & Stryer 2012 Fig 3.34 Matrix-assisted Laser desorption/ionization –time of flight MALDI-TOF • Permits the ionization of proteins that formerly could not be efficiently ionized due to their high MW and low volatility. • Laser vaporizes solvent – some protein enter gas phase too. • Protein ionizes and is separated on a mass/charge ratio. Electrospray ionization mass spectrometry • Peptide in solution sprayed from capillary tube at high voltage to produce highly charged droplets • Solvent soon evaporates to give peptide ions in gas phase • – Yield +0.5 to +2.0 charge per kilodalton from, e.g., Arg & Lys protonation ESI mass spectrum of horse heart apomyolobin • Measures mass/charge (m/z) ratio • Electrospray ionization (ESI) does NOT destroy proteins as earlier mass spec techniques did. • Mass spectrum: series of peaks of ions differing by a single charge and mass of 1 proton. • Each peak corresponds to an m/z ratio of an (M + nH)n+ ion • Can take two adjacent peaks and solve two linear equations to get MW.
Tandem Mass Spectroscopy for Peptide Sequencing Berg, Tymoczko, & Stryer 2012 Fig 3.36 Tandem refers to two mass spectrometers in series Ions of proteins, i.e. precursor ions, from the 1st mass spec are broken into smaller peptide chains, i.e. product ions, by bombardment with atoms of an inert gas. These are in turn passed to a 2nd mass analyzer. • Product ions can be formed such that individual amino acids are cleaved from the precursor ion such that a family of ions can be produced • Each ion represents the original peptide minus one or more amino acids from the end. • The mass difference between the peaks in the plot represent the sequence of the amino acids. “By comparing molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acids can be determined” Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113
Polypeptide Cleavage Endopeptidases Trypsin Rn-1 = pos chg res: Arg, Lys; Rn≠Pro (C-side) Chymotrypsin Rn-1 = bulky hydroph res Phe, Trp, Tyr; Rn≠Pro (C-side) Elastase Rn-1 = small neut. Res: Ala, Gly, Ser, Val; Rn≠Pro Thermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side) Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side) Endopeptidase V8 Rn-1 = Glu Cyanogen Bromide (CNbr) Rn = Met (C side) Endopeptidases hydrolyze internal peptide bonds and are used to fragment polypeptides but require certain adjacent side chains. Voet, Voet, & Pratt 2013 Table 5.4
Edman Degradation of Proteins Voet, Voet & Pratt 2013 Figure 5.16 Use repeated (sequential) cycles of the Edman degradation. Trifluoroacetic acid cleavage of the N-terminal amino acid does NOT hydrolyze the other peptides bonds. Identify PTH-amino acid by chromatographic techniques.
Protein Sequence Determination using Overlapping Fragments Voet, Voet & Pratt 2013 Figure 5.119
Determining Disulfide Bond Location(s) Voet, Voet & Pratt 2013 Figure 5.20
Sites to Find Sequence Data Voet, Voet & Pratt 2013 Fig 5.21 Voet, Voet & Pratt 2013 Table 5.5
Cytochrome c Sequence Analyses Voet, Voet & Pratt 2013 Table 5.6
Cytochrome c Phylogenetic Tree Voet, Voet & Pratt 2013 Figure 5.22
Protein Evolution, Gene Duplication & Protein Modules Protein evolution rates The rate at which mutations are incorporated into a protein are dependent on the degree to which a change in an amino acid effects a protein’s function Gene duplication Proteins with similar functions tend to have similar sequences. New related function can arise by gene duplication. An aberrant genetic recombination in which one chromosome acquires both copies of a primordial gene. Protein modules New proteins (and functions) can also be generated by incorporation of various 40-100 amino acid module or motifs.
Sample Protein Evolution Rates Voet, Voet & Pratt 2013 Figure 5.24 Proteins mutate at different rates over time. But mutations in the DNA typically occur at the same rate Differences due to the rate at which functionally or structurally acceptable changes occurs. That is those changes that are NONLETHAL
SECONDARY STRUCTURE The local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformation of its side chains.
Levels of Protein Structure Voet, Voet & Pratt 2013 Figure 6.1
The (trans) Peptide Bond - Structure Voet, Voet & Pratt 2013 Figure 6.2 In most cases in the protein backbone the peptide bond is in the trans configuration Bond angles and lengths effect to a large extent the freedom of movement and the configuration of the protein. (Important!) Means -carbons of adjacent amino acids are on opposite sides So less steric hindrance of adjacent amino acids side chains Find ~ 8 kJ greater stability of the trans vs the cis configuration Resonance give rise to 40% double bond character
Polypeptide: Extended Conformation & Torsion Angles Voet, Voet & Pratt 2013 Figure 6.3; 6.4 “peptide group” Definition:  and  = 180º when the polypeptide chain is fully extended. They increase clockwise when looking from Cα
Peptide Bonds: Steric Interference Amide hydrogen Carbonyl oxygen Steric interferenceVoet, Voet & Pratt 2013 Figure 6.5
Ramachandran Diagram (Allowed Bond Angles ) Sterically allowed angles for all aa except Gly & Pro α-helix α-helix (left handed) ↑↑ -pleated sheet Note: Gly is less sterically hindered van der Waals radii, the attractive and repulsive forces we covered in earlier lectures have a significance for protein structure. Voet, Voet & Pratt 2013 Figure 6.6
Protein α-Helix Structure Voet, Voet & Pratt 2013 Figure 6.7 Right-handed helix rightleft Lehninger 2000 Box 6.1 5.4Å H-bond every 4th residue Amide H Carbonyl O The alpha helix structures is one of the most stable and is therefore one of the most abundant biological structures. Helix core is tightly packed such that the atoms are at or near their van der Waals radii. The carbonyl oxygen on residue N is hydrogen bonded to the amide hydrogen on residue N+4 an optimum bond length of 2.8Å. This is a source of great thermodynamic stability.
-Helix Stability and Amino Acid Sequence Interactions between amino acids can stabilize or destabilize the helix. •e.g. a long block of Glu residues will not form an -helix at pH 7.0 due to the negatively charged carboxyl groups overpowering H-bonds •Many adjacent Lys and/or Arg residues with pos. charges will repel each other at pH 7.0 •The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close together in the backbone sequence •The twist of the helix ensures that critical interactions occur between a side chain (R-group) and another 3 or 4 residues away. •Positively charged amino acids are often found three residues away from a negatively charge amino acid – supports ion-pair formation •Aromatic residues are often 3 residues apart to support hydrophobic interactions. •Proline (N in rigid ring structure) causes a kink in -helix. Rarely found in helix
Constraints on -Helix Stability (Summary) 1. Electrostatic repulsion or attraction between successive amino acids with charged R groups. 2. Bulkiness of adjacent R groups. 3. Interactions between amino acid side chains spaced 3 (or 4) residues apart. 4. The occurrence of Pro or Gly residues. 5. Interaction between amino acid residues at the ends of the helical segment and the inherent electric dipole of the helix.
Electric Dipole of the Peptide Bond & Interactions Between - Helix Residues Three Apart Lehninger 2000 Figure 6.6 Arg103 side chain Asp100 side chain Lehninger 2000 Figure 6.5 Troponin c protein segment amino carbonyl The electric dipole of the peptide bond is transmitted along an -helical segment via the intrachain hydrogen bonds and this results in an overall helix dipole.
Protein -Sheet Structures Pleated -Sheet -Sheets: parallel vs Antiparallel Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11 Space-filling Antiparallel -Sheet -sheet makes full use of the hydrogen bonding capacity of the polypeptide backbone H-bonding occurs between neighboring polypeptide chains, i.e. interchain, rather than intrachain.
Historical Classification of Proteins Globular polypeptide chains folded in to spherical or globular shape. These often contain several types of secondary structure. Typically most enzymes and regulatory proteins. Fibrous polypeptide chains arranged in long chains or sheets. Usually consist of a single type of secondary structure. Typically provide support, shape and external protection to vertebrates. Alberts et al 2004 Fig 4.9
Fibrous Proteins Fibrous proteins share properties that convey strength and/or flexibility to structures in which they are part. In each case the fundamental structural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water as a result of the high concentration of hydrophobic residues on the protein surface and interior. The hydrophobic residues are largely buried via packing many similar polypeptides chains together to form elaborate supramolecular complexes.
-Keratin Structure: coiled coil Voet, Voet & Pratt 2013 Figure 6.15 A “Permanent Wave” Lehninger 2000 Box 6.2 Rich in Ala, Val, Leu, Ile, Met and Phe – hydrophobic residues A coiled coil - composed of two parallel - helices that are twisted around each other to form a left-handed supertwisted coiled coil.
Silk fibroin -Sheets in Side View Voet, Voet & Pratt 2002 Figure 6.16 Typical repeat: (Gly-Ser-Gly-Ala-Gly-Ala)n Gly Ala or Ser •Has great strength •Not very extensible (would break polypeptide chain covalent bonds) •Very flexible (Neighboring sheets associate with weak van der Waals forces).
Collagen Triple Helix Structure Voet, Voet & Pratt 2013 Figure 6.17 Composition: ~33% Gly ~15-30% Pro, Hyp and Hyl Repeating Sequence: Gly-X-Y where: X is often Pro, Y is often Hyp. Hyl is sometimes at Y Hyp = 4-hydroxyprolyl Hyl = 5-hydroxylysyl •most abundant vertebrate protein occurring •fibers form the major stress bearing components of connective tissues •Three parallel, left- handed helical polypeptide chains with three residues per turn twisted together to form a right-handed superhelical structure.
Collagen’s Molecular Interactions Voet, Voet & Pratt 2002 Figure 6.18 Space-filling model H-bonding in collagen triple helix H-Bond Voet, Voet & Pratt 2013 Figure 6.18a Ball & stick model Every third polypeptide residue passes through the very crowded center of the superhelix, hence the repeated gly every third residue.
Lehninger 2000 Table 6.1 Secondary Structure & Properties Table
Nonrepetitive Protein Structure Native, folded proteins can have nonrepetitive structures that are also ordered like helices or - sheets but they are irregular and therefore more difficult to give a clear, simple description Globular proteins (majority of proteins in nature) can contain a number of secondary structure types. They may have these irregular structures in addition to coils and sheets. The appearance of certain residues outside an α-helix or β-sheet may be nonrandom. Helix capping: Asn and Gln often flank the ends of an α-helix since their side chains can fold back to H-bond with the 4 terminal residues of the helix. β-bulge: a distortion in a β-sheet where a polypeptide strand may have an extra, non H-bonded residue which produces a structural distortion.
 Loop almost always located on the protein surface. May be involved in recognition processes. Turn & Loop Structures in Polypeptides Reverse turn types  Loop in space-filling model Voet, Voet & Pratt 2006 Figure 6.20 found in most proteins with 60 are more residues and are composed on 6 to 16 residues.Voet, Voet & Pratt 2013 Figure 6.14
Lehninger 2005 Figure 4.8 Structure of -turns oxygen hydrogen Type II always Gly at 3 •Connecting elements that link successive runs of an alpha helix or a beta sheet. •A 180° turn of four amino acids •Most common type of turn
Relative Probability of an AA Being in These Secondary Structures Lehninger 2005 Figure 4.10(for illustrative, informational purposes only) Take home message: chemical and physical characteristics of an amino acid (charge, bond angles, etc.) influence its ability to participate in particular secondary structures
TERTIARY STRUCTURE The three-dimensional structure of an entire polypeptide including its side chains Tertiary structure describes the folding of the protein’s secondary structure elements and also specifies the position of each atom in the protein.
Lehninger 2005 Figure 4.16 Myoglobin Tertiary Structure: View Types ribbon mesh Surface contour Ribbon w/ side chains Space- filling w/ side chains Myoglobin is composed of eight relatively straight alpha helices interrupted by bends and some of these are beta turns.
Protein 3-D structure & X-ray crystallography Voet, Voet & Pratt 2013 Figure 6.20b X-ray diffraction pattern of sperm whale myoglobin crystal 3-D electron density of human rhino virus crystal Protein crystal: flavodoxin from Desulfovbrio vulgaris Voet, Voet & Pratt 2013 Figure 6.21 Voet, Voet & Pratt 2002 Figure 6.23
Lehninger 2005 Box 4.4 3-D Protein Structure Determination X-Ray Crystallography •Generate a good protein crystal (not easy). •Detector “sees” a pattern of spots called reflections from X-ray beam. EACH atom makes a contribution to EACH spot. •Massive calculations to produce an electron density map. Nuclei have greatest density. •Yields map of structure
Myoglobin, Globular Proteins, & Tertiary Structure •Positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactions •A dense hydrophobic core is typical of globular proteins. •In dense, closely packed environment weak interactions, e.g. van der Waals, strengthen and reinforce one another.
Lehninger 2005 Box 4.4 Fig 2 NMR in Protein Structure Determination 1-D 2-D #1 Only certain atoms such as 1H, 13C, 15N, 19F, and 31P give rise to an NMR signal. NMR used to identify nuclei and their immediate chemical environment. Also use NOE signals provide information about the distance between atoms
Voet, Voet & Pratt 2013 Fig 6.25 NMR in Protein Structure Determination of Full Structure from 2D Spectrum Backbone showing possible constraints Part of reason for the multiple structures shown is that proteins are dynamic molecules with molecular vibrations occurring in solution. Src protein SH3 domain – 64 residue polypeptide 20 possible structures shown w/ backbone in white
Protein Structural Motifs and Domains In globular proteins the amino acid side chains are distributed according to their polarities to achieve the most energetically favorable conditions. 1. Val, Leu, Ile, Met & Phe occur mostly in the protein interior away from aqueous solvent molecules. 2. Arg, His, Lys, Asp, & Glu are typically located at the proteins surface where their charges can be solvated 3. Ser, Thr, Asn, Gln, & Tyr (uncharged polar) are found on the protein surface but also in the protein’s interior where they are almost always hydrogen bonded.
Side Chain Locations Seen in Space-filling Models Voet, Voet & Pratt 2008 Figure 6.26 α-Helix -Sheet Nonpolar side chains Polar side chains back bone -sheet interior this side sperm whale myoglobin concanavalin A
Voet, Voet & Pratt 2013 Figure 6.27 The hydrophobic side chains are in orange and are closer to the protein’s interior and near the porphyrin ring. Horse Heart Cytochrome c Structure Hydrophillic side chains Hydrophobic side chains Fe atom & heme Hydrophilic sides chains are shown in green and can be seen to be at the protein’s surface.
Protein Motifs (Supersecondary Structures) There are grouping of certain secondary structural elements that occur in many unrelated globular proteins. •Most common motif is an -helix connecting two parallel strands of a -sheet.  motif •Antiparallel strands connected by relatively tight reverse turns  hairpin •Two successive antiparallel helices pack against each other with their axis inclined  motif. •Extended  sheet can role up to form three different types of barrels.  barrel
Protein Structural Motifs α   hairpin α α  barrels Voet, Voet & Pratt 2008 Figure 6.28 Different proteins combine these structures in various ways to achieve their function. Certain successful and energetically favorable designs are preserved in many diverse proteins.
The Rossman Fold Nucleotide binding site Voet, Voet & Pratt 2002 Figure 6.29 A prime example of a structure function- relationship Binds dinucleotides such as NAD+. Utilizes  strands which form a parallel sheet with -helical connections Two such  units are shown. Voet, Voet & Pratt 2013 Figure 6.31
Protein Domains Polypeptide chains > ~200 residues usually fold into two or more globular clusters. • Typical domain 100 – 200 residues with an 25 Å avg. diameter. • Neighboring domains are usually connected by one or two polypeptide segments. Many domains are structurally independent units with characteristics of a small globular protein • Domains generally consist of two or more layers of secondary structures – seals off a domain’s hydrophobic core from aqueous environment. • Domains often have a specific function, e.g. nucleotide binding.
Domains in Evolution 1. Form stable folding patterns. 2. Tolerate amino acid deletions, substitutions, & insertions which makes them better able to survive evolutionary changes. 3. Support essential biological functions. Common protein structures likely arose and persisted because of their ability to: Studies of proteins support the concept that essential structural and functional elements of proteins rather than their amino acid residues are conserved during evolution, e.g. changes in like residues that do not appreciably change structure are not dysfunctional.
QUATERNARY STRUCTURE The spatial arrangement of the subunits of a multisubunit protein •Subunits typically associate via noncovalent interactions. •Contact regions between subunits resemble the interior of single subunit proteins. Definitions: Oligomer – proteins with more than one subunit Protomer – repeating structural subunits of a protein Design Benefits: •Easier to synthesize multiple smaller subunits than one large polypeptide chain free from error. •A subunit with an error can more easily replaced than a single large polypeptide – more efficient.
Quaternary Structure of Hemoglobin Voet, Voet & Pratt 2008 Figure 6.33 •An oligomeric protein •Each of the four subunits 1212 is shown in a different color. (Heme is red)
Oligomeric Protein Symmetry Examples Voet, Voet & Pratt 2013 Figure 6.34 Proteins can only have rotational symmetry Related by single axis of rotation When n-fold rotation axis intersects a 2- fold rotation axis at 90° Other types of symmetry based on geometrical objects
Protein Folding & Stability •Normally for biological structures, the molecules exist in conformations that are at energy minimums, i.e. the most thermodynamically stable. •Hydrophobic effects, electrostatic interactions, and hydrogen bonding, the noncovalent interactions, each provide energies of thousands of kJ/mol over an entire protein. •Thermodynamic studies of native proteins revealed that native protein are only marginally stable under physiological conditions as the free energy required to denature them is approximately 0.4 kcal/mol per amino acid residue. For 100 residue protein ONLY 40kJ/mol more stable than unfolded Conclude: a protein’s structure is in fact a delicate balance of counteracting forces.
Stabilizing Forces Hydrophobic effect - Nonpolar molecules seek thermodynamically (entropically) to minimize their contact with water. This is the major determinant of native protein structure – greatest contribution to stability
Hydropathy Plot Voet, Voet & Pratt 2008 Figure 6.35 Protein interior Protein exterior Plot combines hydrophobic and hydrophilic tendencies of individual amino acids. The greater a side chain’s hydropathy the more like it is to be in a protein’s interior.
Electrostatic Interactions The association of two ionic protein groups of opposite charge is called an ion pair or salt bridge. Approximately 75% of charged residues are involved in ion pairs and are mostly on the protein surface.
Chemical Cross-Linking Disulfide Bonds Thought that disulfide bonds are not so much a stabilizing force, but they may function to lock in a particular conformation of a polypeptide backbone. Since the cell cytoplasm is a reducing environment most intracellular proteins do not have disulfide bridges. Metal Ions can also internally link proteins
Electrostatic Contributions to Protein Stability •Van der Waals interactions are an important stabilizing force in the closely packed protein interior where they act over short distances and are lost when the protein is unfolded. •Hydrogen bonds make a minor contribution because those groups can H-bond with water as a protein unfolds. H-bonds do “select” the unique native structure of a protein from a small number of hydrophobically stabilized conformations. •Ions pairs also make minor contributions because the free energy of the pair does not compensate for the loss of side chain entropy and the loss of the free energy of solvation.
Protein Denaturation/Renaturation Proteins can be denatured by a variety of conditions & substances. Heating (adding energy) Most proteins exhibit a sharp transition over a narrow temperature range indicative of the protein unfolding in a cooperative manner. pH variation Alters the ionization of amino acid side chains which results in changes in charge distributions and H-bonding needs. Detergents (amphipathic molecules) Associated with nonpolar residues and disrupt the hydrophobic interactions Chaotropic agents (urea, guanidinium chloride) Ions or small organic molecules that at high 5- 10M concentration disrupt hydrophobic interactions
Lehninger 2005 Figure 4.26 Protein Denaturation Curves
Protein Renaturation Anfinsen classic experiment with RNAse A (4 disulfide bonds must reform): Proteins can spontaneously fold into their native conformation under physiological conditions – protein’s primary structure dictates its three dimensional structure.
Protein Denaturation/Renaturation Voet, Voet & Pratt 2008 Figure 6.39
Thermodynamics of Protein Folding Lehninger 2000 Figure 6.27 Number of possible conformations is large and therefore the conformation energy is large. At this point only a small percentage of the intramolecular interactions found in the native state are present Decreasing number of possible states, i.e. the entropy Amount of protein in native state increasing Free energy is decreasing as we head to a free energy minimum at the bottom, i.e., native state. Energy max Energy min native min
Protein Misfolding: Prions & Disease Lehninger 2000 Box 6.4 •Brain becomes riddled with holes •Caused by a single 28 kd protein called a prion protein •Normal Prp is mostly alpha helical. •Illness occurs when an altered form of Prp called PrpScr is present which has mixed alpha helix and beta sheets Prion: proteinaceous infectious particle
Alzheimers and -Amyloid Protein -Amyloid protein is normally present in the human brain but its function is unknown In Alzheimers a 40 residue (shorter) segment of this protein forms fibrous deposits or plaques. This cleaved protein tends to aggregate only with itself. This protein does not fold properly! Amyloid plaques w/ Aβ protein in brain tissue of human Alzheimers patient Native (N) <=> Unfolded (U) Keq = [U]/[N] = e-∆G˚’/RT As the ∆G˚’ for unfolding decreases the portion unfolded proteins increases.
Proteins that Help Native Protein Folding Molecular chaperones – proteins that bind to unfolded and partially folded polypeptide chains to prevent improper folding by prevent improper association of hydrophobic regions that could lead to polypeptide aggregation, precipitation, or non-native folding. Important for multisubunit and multidomain proteins. First described as heat shock proteins (Hsp) Most are ATPases Voet, Voet & Pratt 2012 Figure 6.45
Proteins are Dynamic Structures Myoglobin Voet, Voet & Pratt 2013 Figure 6.39 •Remember that proteins are in fact dynamic molecules with normal structural fluctuations •These fluctuations are important for function particularly in enzymes. •Snapshots are for the structure of myoglobin seen over 4 x 10-12 seconds
End of Lectures

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Protein structure

  • 1.
    Protein Structure Student Edition5/23/13 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall chazotte@campbell.edu Web Site: http://www.campbell.edu/faculty/chazotte Original material only ©2004-14 B. Chazotte
  • 2.
    Goals •Understand the basesof & differences between primary, secondary, tertiary, & quaternary protein structure. •Be familiar with basic protein purification/sequencing methods & how they depend on the physical & chemical properties of proteins. •Understand the physical and chemical forces that determine secondary structure, including the peptide bond. Learn the basic types of secondary structure: -helix, -sheet, random coil & non-repetitive structures. •Understand the physical & chemical forces that determine tertiary structure - protein domains and motifs. •Be acquainted with techniques like NMR & X-ray crystallography that help determine protein structure. •Understand the physical and chemical forces that determine quaternary structure – protein folding, denaturation, renaturation, hydropathy plots. •Remember how protein structure and structural changes reflect the influence of thermodynamic concepts on structural stability.
  • 3.
    Levels of ProteinStructure •Primary •Secondary •Tertiary •Quaternary Refer to the three- dimensional shape of folded polypeptide chains
  • 4.
    Protein Diversity For aprotein of n residues there are 20n possible sequences For 40 residue protein 1.1 x 1052 For 100 residue protein 1.27 x 10130 Definitions •Peptides – typically less than 40 residues Dipeptide: 2 amino acids; Tripeptide: 3 amino acids Oligopeptide: many amino acids •Proteins – typically polypeptides with 40 or more residues •Multisubunit proteins - proteins with several identical or nonidentical subunits.
  • 5.
    PRIMARY STRUCTURE & ANALYSIS Primary Structure:the amino acid sequence of a protein’s polypeptide chain or chains. Sometimes referred to as the covalent structure.
  • 6.
    Conjecture on theLimitations of Protein Size Minimum: 40 residues – near the limit for a polypeptide to be able to fold into a discrete stable shape that permits it to carry out its basic function. Maximum: ~1000 residues – near the limit for the protein synthetic machinery to produce a peptide with minimal errors in the polypeptide, mRNA template, or gene DNA.
  • 7.
    Logic of AminoAcid Sequences in Proteins The characteristics of a protein depend more on the sequence of amino acids rather than its composition. The presence of an amino acid with its characteristic physical & chemical properties at a particular place in a protein influences the protein’s properties. (review Amino Acids lecture) The 3-D shape of a protein is a consequence of the intermolecular forces among its various residues. (review Chemical Bonding lecture) Voet, Voet & Pratt 2013 Chap. 5.1
  • 8.
    Primary Structure ofBovine Insulin Voet, Voet & Pratt 2013 Figure 5.1
  • 9.
    Studying Proteins byIsolating Them
  • 10.
    Protein Purification Crude Extract– 1st step whether protein is from tissue or microbe, break open the cell and release the proteins into solution. Fractionation – step where proteins are separated into different fractions bases on some chemical or physical property such as size or charge. May utilize protein solubility i.e. (pH), salt concentration, temperature, etc.
  • 11.
    Proteins Must beStabilized after Isolation Care must be taken to preserve protein structure and function after its is removed from its natural environment were it was stable. •pH To prevent denaturation (loss of structure) or function proteins are placed in buffered solutions at or near their native pH. •Temperature Protein purification is normally carried out at low temperature ~0º C. While some proteins are thermally stable at high temperatures, others may be affected by temperature a few degrees higher than the native environment. •Degradative Enzymes During isolation various nucleases and proteases are released from their places in the cell and can degrade nucleic acids or proteins unless temperature, pH or inhibitory agents are added. •Adsorption to Surfaces Solutions are handled to minimize foaming and are kept concentrated as interfaces (air-water, glass, plastic) can cause denaturation. •Storage To maintain protein stability. Cold (-70º C or -196º C liq N2), sometime under N2(g) to remove oxygen and prevent slow oxidation. Some of the goals are to minimize microbial growth and/or oxidation. Voet, Voet & Pratt 2013 p.96
  • 12.
    Assay of PurifiedProteins To purify a protein it is necessary to measure how much you have  need a specific assay. •Easier for enzymes as they produce a product proportional to the amount of enzyme present. •Colored or fluorescent products are especially helpful •Can also use a coupled enzyme reaction, i.e. 2nd enzyme •Can use immunochemical assays.
  • 13.
  • 14.
    Some Separation Techniques ChargeIon Exchange Chromatography Electrophoresis Polarity Hydrophobic Interaction Chromatography Size Gel Filtration Chromatography SDS-Polyacrylamide Electrophoresis Ultracentrifugation Binding Specificity Affinity Chromatography These separation techniques utilize differences in the physical and/or chemical properties that arise from the differences amino acid composition.
  • 15.
    Protein Fractionation bySalting Out Voet, Voet & Pratt 2013 Figure 5.5 Protein solubility depends on: •Concentration of dissolved salts •Solvent polarity •pH •Temperature By careful manipulation of these properties it is possible to selectively precipitate out certain proteins and leave the other soluble.
  • 16.
    Protein Separation byIon Exchange Chromatography Voet, Voet & Pratt 2013 Figure 5.6 Ion exchange chromatography makes use of the fact that opposite charges attract Polyelectrolytes such as proteins that have both negative and positive charges will bind to cation or anion exchangers depending on the protein’s net charge The binding affinity (Strength of binding) depends on the presence of other ions that compete with the protein for binding sites on the immobile phase and the pH which in terms effects the protein’s net charge. Anion exchanger: e.g., DEAE Matrix–CH2-CH2-NH(CH2CH3)2 + Cation exchanger: e.g., CM Matrix-CH2COO-
  • 17.
    Protein Separation byGel Filtration Chromatography Voet, Voet & Pratt 2013 Figure 5.7 A bead can have different pore sizes (holes) depending on the extent of cross-linking in its component polymer. The larger proteins that are excluded from the beads have a shorter path and leave the column sooner.
  • 18.
    Protein Separation byAffinity Chromatography Voet, Voet & Pratt 2013 Figure 5.8 Utilize the ability of certain proteins (via biochemical properties) that are able to bind specific molecules non-covalently. Bind a specific molecule called a ligand to an inert matrix – immobile phase Column conditions are then changed, e.g. pH, ionic strength or high ligand concentration, to permit the protein to elute in a highly purified form.
  • 19.
    SDS-PAGE of Supernatants& Membrane Fraction from a Bacterium Voet, Voet & Pratt 2006 Figure 5.9 & 2013 Fig 5.9 & 5.10 Gel electrophoresis - a molecular sieving approach SDS, sodium dodecyl sulfate, when added to a protein solution binds 1 molecule of SDS per two amino acids or 1.4g per g protein.
  • 20.
    Separation by Zonal Centrifugation Voet,Voet & Pratt 2013 Figure 5.12 Gradient preparation Berg, Tymoczko, & Stryer 2012 Fig 3.16
  • 21.
    Protein Sequencing 1. Sequenceis a prerequisite for determining protein’s 3-D structure and understanding its molecular mechanism. 2. Sequence comparisons among analogous proteins from different species yield insights into protein function as well as reveal evolutionary relationships among proteins 3. Many inherited diseases are caused by point mutations in the amino acid sequence. Sequence analysis can assist with diagnostic testing and therapy development. Voet, Voet & Pratt 2006 Chapter 5 A protein must be broken down into fragments small enough to be individually sequenced. The fragments are used to reconstruct the protein by analyzing the fragment overlaps.
  • 22.
    Sanger Method forProtein Sequencing Voet, Voet & Pratt 2013 p.108 Box 5.1 Bind to terminal amino groups to form a yellow dinitrophenyl derivative Hydrolyze protein Identify terminal amino acid chromatographically Also today nucleic acids sequencing is frequently used to determine protein sequences
  • 23.
    Basic Logic ofProtein Sequencing Voet, Voet & Pratt 2013 Figure 5.13 1. polypeptide chains that are linked by disulfide bonds are separated by reduction of the sulfhydral groups of cysteine. 2. chemical or enzymatic means are used to cleave the resultant polypeptide chains into smaller fragments. 3. Each small fragment is sequenced. 4. Compare overlapping sequences produced by different enzymes or chemical degradations to logically reconstruct original protein sequence 5. Repeat process without cleaving the disulfide bonds to determine where those bonds are located
  • 24.
    End Group Analysis Usedto determine the number of distinct polypeptide chains in a protein (if end groups not chemically blocked). •There are several procedures for the N-terminus. •No reliable chemical procedure for C-terminus, an enzymatic approach uses carboxypeptidases.
  • 25.
    Protein Sequencing End GroupAnalysis with Danzyl Chloride Voet, Voet & Pratt 2013 Figure 5.14 Conjugate the fluorophore to primary amine(s). Perform an acid hydrolysis Identify via chromatography the labeled amino acid.
  • 26.
    Disulfide Bond Cleavage Needto cleave to separate polypeptide chains. Two methods: • oxidative cleavage with performic acid disadvantage: destroys met and Trp indol side chain • reductive cleavage with mercaptan e.g. 2-mercaptoethanol. Usually alkylate product with iodoacetic acid to prevent disulfide bond reformation.
  • 27.
    Peptide Hydrolysis Chemical approach Acidhydrolysis disadvantages – destroys Ser, Thr, Tyr & Trp converts Asn & Gln to Asp & Glu, respectively Base Hydrolysis disadvantages – destroys Cys, Ser, Thr & Arg Biochemical Approach Enzymatic Hydrolysis disadvantages – often incomplete some autodigestion
  • 28.
    Amino Acid Analysis Separationby HPLC Voet, Voet & Pratt 2006 Figure 5.15 Complete hydrolysis will yield the composition but not the sequence
  • 29.
    Molecular Mass Determination byMass Spectrometry Voet, Voet, & Pratt 2013 Fig 17a,bBerg, Tymoczko, & Stryer 2012 Fig 3.34 Matrix-assisted Laser desorption/ionization –time of flight MALDI-TOF • Permits the ionization of proteins that formerly could not be efficiently ionized due to their high MW and low volatility. • Laser vaporizes solvent – some protein enter gas phase too. • Protein ionizes and is separated on a mass/charge ratio. Electrospray ionization mass spectrometry • Peptide in solution sprayed from capillary tube at high voltage to produce highly charged droplets • Solvent soon evaporates to give peptide ions in gas phase • – Yield +0.5 to +2.0 charge per kilodalton from, e.g., Arg & Lys protonation ESI mass spectrum of horse heart apomyolobin • Measures mass/charge (m/z) ratio • Electrospray ionization (ESI) does NOT destroy proteins as earlier mass spec techniques did. • Mass spectrum: series of peaks of ions differing by a single charge and mass of 1 proton. • Each peak corresponds to an m/z ratio of an (M + nH)n+ ion • Can take two adjacent peaks and solve two linear equations to get MW.
  • 30.
    Tandem Mass Spectroscopyfor Peptide Sequencing Berg, Tymoczko, & Stryer 2012 Fig 3.36 Tandem refers to two mass spectrometers in series Ions of proteins, i.e. precursor ions, from the 1st mass spec are broken into smaller peptide chains, i.e. product ions, by bombardment with atoms of an inert gas. These are in turn passed to a 2nd mass analyzer. • Product ions can be formed such that individual amino acids are cleaved from the precursor ion such that a family of ions can be produced • Each ion represents the original peptide minus one or more amino acids from the end. • The mass difference between the peaks in the plot represent the sequence of the amino acids. “By comparing molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acids can be determined” Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113
  • 31.
    Polypeptide Cleavage Endopeptidases Trypsin Rn-1= pos chg res: Arg, Lys; Rn≠Pro (C-side) Chymotrypsin Rn-1 = bulky hydroph res Phe, Trp, Tyr; Rn≠Pro (C-side) Elastase Rn-1 = small neut. Res: Ala, Gly, Ser, Val; Rn≠Pro Thermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side) Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side) Endopeptidase V8 Rn-1 = Glu Cyanogen Bromide (CNbr) Rn = Met (C side) Endopeptidases hydrolyze internal peptide bonds and are used to fragment polypeptides but require certain adjacent side chains. Voet, Voet, & Pratt 2013 Table 5.4
  • 32.
    Edman Degradation ofProteins Voet, Voet & Pratt 2013 Figure 5.16 Use repeated (sequential) cycles of the Edman degradation. Trifluoroacetic acid cleavage of the N-terminal amino acid does NOT hydrolyze the other peptides bonds. Identify PTH-amino acid by chromatographic techniques.
  • 33.
    Protein Sequence Determination usingOverlapping Fragments Voet, Voet & Pratt 2013 Figure 5.119
  • 34.
    Determining Disulfide BondLocation(s) Voet, Voet & Pratt 2013 Figure 5.20
  • 35.
    Sites to FindSequence Data Voet, Voet & Pratt 2013 Fig 5.21 Voet, Voet & Pratt 2013 Table 5.5
  • 36.
    Cytochrome c SequenceAnalyses Voet, Voet & Pratt 2013 Table 5.6
  • 37.
    Cytochrome c PhylogeneticTree Voet, Voet & Pratt 2013 Figure 5.22
  • 38.
    Protein Evolution, Gene Duplication& Protein Modules Protein evolution rates The rate at which mutations are incorporated into a protein are dependent on the degree to which a change in an amino acid effects a protein’s function Gene duplication Proteins with similar functions tend to have similar sequences. New related function can arise by gene duplication. An aberrant genetic recombination in which one chromosome acquires both copies of a primordial gene. Protein modules New proteins (and functions) can also be generated by incorporation of various 40-100 amino acid module or motifs.
  • 39.
    Sample Protein EvolutionRates Voet, Voet & Pratt 2013 Figure 5.24 Proteins mutate at different rates over time. But mutations in the DNA typically occur at the same rate Differences due to the rate at which functionally or structurally acceptable changes occurs. That is those changes that are NONLETHAL
  • 40.
    SECONDARY STRUCTURE The localspatial arrangement of a polypeptide’s backbone atoms without regard to the conformation of its side chains.
  • 41.
    Levels of ProteinStructure Voet, Voet & Pratt 2013 Figure 6.1
  • 42.
    The (trans) PeptideBond - Structure Voet, Voet & Pratt 2013 Figure 6.2 In most cases in the protein backbone the peptide bond is in the trans configuration Bond angles and lengths effect to a large extent the freedom of movement and the configuration of the protein. (Important!) Means -carbons of adjacent amino acids are on opposite sides So less steric hindrance of adjacent amino acids side chains Find ~ 8 kJ greater stability of the trans vs the cis configuration Resonance give rise to 40% double bond character
  • 43.
    Polypeptide: Extended Conformation &Torsion Angles Voet, Voet & Pratt 2013 Figure 6.3; 6.4 “peptide group” Definition:  and  = 180º when the polypeptide chain is fully extended. They increase clockwise when looking from Cα
  • 44.
    Peptide Bonds: StericInterference Amide hydrogen Carbonyl oxygen Steric interferenceVoet, Voet & Pratt 2013 Figure 6.5
  • 45.
    Ramachandran Diagram (Allowed BondAngles ) Sterically allowed angles for all aa except Gly & Pro α-helix α-helix (left handed) ↑↑ -pleated sheet Note: Gly is less sterically hindered van der Waals radii, the attractive and repulsive forces we covered in earlier lectures have a significance for protein structure. Voet, Voet & Pratt 2013 Figure 6.6
  • 46.
    Protein α-Helix Structure Voet,Voet & Pratt 2013 Figure 6.7 Right-handed helix rightleft Lehninger 2000 Box 6.1 5.4Å H-bond every 4th residue Amide H Carbonyl O The alpha helix structures is one of the most stable and is therefore one of the most abundant biological structures. Helix core is tightly packed such that the atoms are at or near their van der Waals radii. The carbonyl oxygen on residue N is hydrogen bonded to the amide hydrogen on residue N+4 an optimum bond length of 2.8Å. This is a source of great thermodynamic stability.
  • 47.
    -Helix Stability andAmino Acid Sequence Interactions between amino acids can stabilize or destabilize the helix. •e.g. a long block of Glu residues will not form an -helix at pH 7.0 due to the negatively charged carboxyl groups overpowering H-bonds •Many adjacent Lys and/or Arg residues with pos. charges will repel each other at pH 7.0 •The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close together in the backbone sequence •The twist of the helix ensures that critical interactions occur between a side chain (R-group) and another 3 or 4 residues away. •Positively charged amino acids are often found three residues away from a negatively charge amino acid – supports ion-pair formation •Aromatic residues are often 3 residues apart to support hydrophobic interactions. •Proline (N in rigid ring structure) causes a kink in -helix. Rarely found in helix
  • 48.
    Constraints on -HelixStability (Summary) 1. Electrostatic repulsion or attraction between successive amino acids with charged R groups. 2. Bulkiness of adjacent R groups. 3. Interactions between amino acid side chains spaced 3 (or 4) residues apart. 4. The occurrence of Pro or Gly residues. 5. Interaction between amino acid residues at the ends of the helical segment and the inherent electric dipole of the helix.
  • 49.
    Electric Dipole of thePeptide Bond & Interactions Between - Helix Residues Three Apart Lehninger 2000 Figure 6.6 Arg103 side chain Asp100 side chain Lehninger 2000 Figure 6.5 Troponin c protein segment amino carbonyl The electric dipole of the peptide bond is transmitted along an -helical segment via the intrachain hydrogen bonds and this results in an overall helix dipole.
  • 50.
    Protein -Sheet Structures Pleated-Sheet -Sheets: parallel vs Antiparallel Voet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11 Space-filling Antiparallel -Sheet -sheet makes full use of the hydrogen bonding capacity of the polypeptide backbone H-bonding occurs between neighboring polypeptide chains, i.e. interchain, rather than intrachain.
  • 51.
    Historical Classification ofProteins Globular polypeptide chains folded in to spherical or globular shape. These often contain several types of secondary structure. Typically most enzymes and regulatory proteins. Fibrous polypeptide chains arranged in long chains or sheets. Usually consist of a single type of secondary structure. Typically provide support, shape and external protection to vertebrates. Alberts et al 2004 Fig 4.9
  • 52.
    Fibrous Proteins Fibrous proteinsshare properties that convey strength and/or flexibility to structures in which they are part. In each case the fundamental structural unit is a simple repeating element of secondary structure. All fibrous proteins are insoluble in water as a result of the high concentration of hydrophobic residues on the protein surface and interior. The hydrophobic residues are largely buried via packing many similar polypeptides chains together to form elaborate supramolecular complexes.
  • 53.
    -Keratin Structure: coiledcoil Voet, Voet & Pratt 2013 Figure 6.15 A “Permanent Wave” Lehninger 2000 Box 6.2 Rich in Ala, Val, Leu, Ile, Met and Phe – hydrophobic residues A coiled coil - composed of two parallel - helices that are twisted around each other to form a left-handed supertwisted coiled coil.
  • 54.
    Silk fibroin -Sheetsin Side View Voet, Voet & Pratt 2002 Figure 6.16 Typical repeat: (Gly-Ser-Gly-Ala-Gly-Ala)n Gly Ala or Ser •Has great strength •Not very extensible (would break polypeptide chain covalent bonds) •Very flexible (Neighboring sheets associate with weak van der Waals forces).
  • 55.
    Collagen Triple HelixStructure Voet, Voet & Pratt 2013 Figure 6.17 Composition: ~33% Gly ~15-30% Pro, Hyp and Hyl Repeating Sequence: Gly-X-Y where: X is often Pro, Y is often Hyp. Hyl is sometimes at Y Hyp = 4-hydroxyprolyl Hyl = 5-hydroxylysyl •most abundant vertebrate protein occurring •fibers form the major stress bearing components of connective tissues •Three parallel, left- handed helical polypeptide chains with three residues per turn twisted together to form a right-handed superhelical structure.
  • 56.
    Collagen’s Molecular Interactions Voet,Voet & Pratt 2002 Figure 6.18 Space-filling model H-bonding in collagen triple helix H-Bond Voet, Voet & Pratt 2013 Figure 6.18a Ball & stick model Every third polypeptide residue passes through the very crowded center of the superhelix, hence the repeated gly every third residue.
  • 57.
    Lehninger 2000 Table6.1 Secondary Structure & Properties Table
  • 58.
    Nonrepetitive Protein Structure Native,folded proteins can have nonrepetitive structures that are also ordered like helices or - sheets but they are irregular and therefore more difficult to give a clear, simple description Globular proteins (majority of proteins in nature) can contain a number of secondary structure types. They may have these irregular structures in addition to coils and sheets. The appearance of certain residues outside an α-helix or β-sheet may be nonrandom. Helix capping: Asn and Gln often flank the ends of an α-helix since their side chains can fold back to H-bond with the 4 terminal residues of the helix. β-bulge: a distortion in a β-sheet where a polypeptide strand may have an extra, non H-bonded residue which produces a structural distortion.
  • 59.
     Loop almostalways located on the protein surface. May be involved in recognition processes. Turn & Loop Structures in Polypeptides Reverse turn types  Loop in space-filling model Voet, Voet & Pratt 2006 Figure 6.20 found in most proteins with 60 are more residues and are composed on 6 to 16 residues.Voet, Voet & Pratt 2013 Figure 6.14
  • 60.
    Lehninger 2005 Figure4.8 Structure of -turns oxygen hydrogen Type II always Gly at 3 •Connecting elements that link successive runs of an alpha helix or a beta sheet. •A 180° turn of four amino acids •Most common type of turn
  • 61.
    Relative Probability ofan AA Being in These Secondary Structures Lehninger 2005 Figure 4.10(for illustrative, informational purposes only) Take home message: chemical and physical characteristics of an amino acid (charge, bond angles, etc.) influence its ability to participate in particular secondary structures
  • 62.
    TERTIARY STRUCTURE The three-dimensionalstructure of an entire polypeptide including its side chains Tertiary structure describes the folding of the protein’s secondary structure elements and also specifies the position of each atom in the protein.
  • 63.
    Lehninger 2005 Figure4.16 Myoglobin Tertiary Structure: View Types ribbon mesh Surface contour Ribbon w/ side chains Space- filling w/ side chains Myoglobin is composed of eight relatively straight alpha helices interrupted by bends and some of these are beta turns.
  • 64.
    Protein 3-D structure& X-ray crystallography Voet, Voet & Pratt 2013 Figure 6.20b X-ray diffraction pattern of sperm whale myoglobin crystal 3-D electron density of human rhino virus crystal Protein crystal: flavodoxin from Desulfovbrio vulgaris Voet, Voet & Pratt 2013 Figure 6.21 Voet, Voet & Pratt 2002 Figure 6.23
  • 65.
    Lehninger 2005 Box4.4 3-D Protein Structure Determination X-Ray Crystallography •Generate a good protein crystal (not easy). •Detector “sees” a pattern of spots called reflections from X-ray beam. EACH atom makes a contribution to EACH spot. •Massive calculations to produce an electron density map. Nuclei have greatest density. •Yields map of structure
  • 66.
    Myoglobin, Globular Proteins,& Tertiary Structure •Positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactions •A dense hydrophobic core is typical of globular proteins. •In dense, closely packed environment weak interactions, e.g. van der Waals, strengthen and reinforce one another.
  • 67.
    Lehninger 2005 Box4.4 Fig 2 NMR in Protein Structure Determination 1-D 2-D #1 Only certain atoms such as 1H, 13C, 15N, 19F, and 31P give rise to an NMR signal. NMR used to identify nuclei and their immediate chemical environment. Also use NOE signals provide information about the distance between atoms
  • 68.
    Voet, Voet &Pratt 2013 Fig 6.25 NMR in Protein Structure Determination of Full Structure from 2D Spectrum Backbone showing possible constraints Part of reason for the multiple structures shown is that proteins are dynamic molecules with molecular vibrations occurring in solution. Src protein SH3 domain – 64 residue polypeptide 20 possible structures shown w/ backbone in white
  • 69.
    Protein Structural Motifsand Domains In globular proteins the amino acid side chains are distributed according to their polarities to achieve the most energetically favorable conditions. 1. Val, Leu, Ile, Met & Phe occur mostly in the protein interior away from aqueous solvent molecules. 2. Arg, His, Lys, Asp, & Glu are typically located at the proteins surface where their charges can be solvated 3. Ser, Thr, Asn, Gln, & Tyr (uncharged polar) are found on the protein surface but also in the protein’s interior where they are almost always hydrogen bonded.
  • 70.
    Side Chain LocationsSeen in Space-filling Models Voet, Voet & Pratt 2008 Figure 6.26 α-Helix -Sheet Nonpolar side chains Polar side chains back bone -sheet interior this side sperm whale myoglobin concanavalin A
  • 71.
    Voet, Voet &Pratt 2013 Figure 6.27 The hydrophobic side chains are in orange and are closer to the protein’s interior and near the porphyrin ring. Horse Heart Cytochrome c Structure Hydrophillic side chains Hydrophobic side chains Fe atom & heme Hydrophilic sides chains are shown in green and can be seen to be at the protein’s surface.
  • 72.
    Protein Motifs (Supersecondary Structures) Thereare grouping of certain secondary structural elements that occur in many unrelated globular proteins. •Most common motif is an -helix connecting two parallel strands of a -sheet.  motif •Antiparallel strands connected by relatively tight reverse turns  hairpin •Two successive antiparallel helices pack against each other with their axis inclined  motif. •Extended  sheet can role up to form three different types of barrels.  barrel
  • 73.
    Protein Structural Motifs α  hairpin α α  barrels Voet, Voet & Pratt 2008 Figure 6.28 Different proteins combine these structures in various ways to achieve their function. Certain successful and energetically favorable designs are preserved in many diverse proteins.
  • 74.
    The Rossman Fold Nucleotidebinding site Voet, Voet & Pratt 2002 Figure 6.29 A prime example of a structure function- relationship Binds dinucleotides such as NAD+. Utilizes  strands which form a parallel sheet with -helical connections Two such  units are shown. Voet, Voet & Pratt 2013 Figure 6.31
  • 75.
    Protein Domains Polypeptide chains> ~200 residues usually fold into two or more globular clusters. • Typical domain 100 – 200 residues with an 25 Å avg. diameter. • Neighboring domains are usually connected by one or two polypeptide segments. Many domains are structurally independent units with characteristics of a small globular protein • Domains generally consist of two or more layers of secondary structures – seals off a domain’s hydrophobic core from aqueous environment. • Domains often have a specific function, e.g. nucleotide binding.
  • 76.
    Domains in Evolution 1.Form stable folding patterns. 2. Tolerate amino acid deletions, substitutions, & insertions which makes them better able to survive evolutionary changes. 3. Support essential biological functions. Common protein structures likely arose and persisted because of their ability to: Studies of proteins support the concept that essential structural and functional elements of proteins rather than their amino acid residues are conserved during evolution, e.g. changes in like residues that do not appreciably change structure are not dysfunctional.
  • 77.
    QUATERNARY STRUCTURE The spatialarrangement of the subunits of a multisubunit protein •Subunits typically associate via noncovalent interactions. •Contact regions between subunits resemble the interior of single subunit proteins. Definitions: Oligomer – proteins with more than one subunit Protomer – repeating structural subunits of a protein Design Benefits: •Easier to synthesize multiple smaller subunits than one large polypeptide chain free from error. •A subunit with an error can more easily replaced than a single large polypeptide – more efficient.
  • 78.
    Quaternary Structure of Hemoglobin Voet,Voet & Pratt 2008 Figure 6.33 •An oligomeric protein •Each of the four subunits 1212 is shown in a different color. (Heme is red)
  • 79.
    Oligomeric Protein Symmetry Examples Voet,Voet & Pratt 2013 Figure 6.34 Proteins can only have rotational symmetry Related by single axis of rotation When n-fold rotation axis intersects a 2- fold rotation axis at 90° Other types of symmetry based on geometrical objects
  • 80.
    Protein Folding &Stability •Normally for biological structures, the molecules exist in conformations that are at energy minimums, i.e. the most thermodynamically stable. •Hydrophobic effects, electrostatic interactions, and hydrogen bonding, the noncovalent interactions, each provide energies of thousands of kJ/mol over an entire protein. •Thermodynamic studies of native proteins revealed that native protein are only marginally stable under physiological conditions as the free energy required to denature them is approximately 0.4 kcal/mol per amino acid residue. For 100 residue protein ONLY 40kJ/mol more stable than unfolded Conclude: a protein’s structure is in fact a delicate balance of counteracting forces.
  • 81.
    Stabilizing Forces Hydrophobic effect- Nonpolar molecules seek thermodynamically (entropically) to minimize their contact with water. This is the major determinant of native protein structure – greatest contribution to stability
  • 82.
    Hydropathy Plot Voet, Voet& Pratt 2008 Figure 6.35 Protein interior Protein exterior Plot combines hydrophobic and hydrophilic tendencies of individual amino acids. The greater a side chain’s hydropathy the more like it is to be in a protein’s interior.
  • 83.
    Electrostatic Interactions The associationof two ionic protein groups of opposite charge is called an ion pair or salt bridge. Approximately 75% of charged residues are involved in ion pairs and are mostly on the protein surface.
  • 84.
    Chemical Cross-Linking Disulfide Bonds Thoughtthat disulfide bonds are not so much a stabilizing force, but they may function to lock in a particular conformation of a polypeptide backbone. Since the cell cytoplasm is a reducing environment most intracellular proteins do not have disulfide bridges. Metal Ions can also internally link proteins
  • 85.
    Electrostatic Contributions to ProteinStability •Van der Waals interactions are an important stabilizing force in the closely packed protein interior where they act over short distances and are lost when the protein is unfolded. •Hydrogen bonds make a minor contribution because those groups can H-bond with water as a protein unfolds. H-bonds do “select” the unique native structure of a protein from a small number of hydrophobically stabilized conformations. •Ions pairs also make minor contributions because the free energy of the pair does not compensate for the loss of side chain entropy and the loss of the free energy of solvation.
  • 86.
    Protein Denaturation/Renaturation Proteins can bedenatured by a variety of conditions & substances. Heating (adding energy) Most proteins exhibit a sharp transition over a narrow temperature range indicative of the protein unfolding in a cooperative manner. pH variation Alters the ionization of amino acid side chains which results in changes in charge distributions and H-bonding needs. Detergents (amphipathic molecules) Associated with nonpolar residues and disrupt the hydrophobic interactions Chaotropic agents (urea, guanidinium chloride) Ions or small organic molecules that at high 5- 10M concentration disrupt hydrophobic interactions
  • 87.
    Lehninger 2005 Figure4.26 Protein Denaturation Curves
  • 88.
    Protein Renaturation Anfinsen classicexperiment with RNAse A (4 disulfide bonds must reform): Proteins can spontaneously fold into their native conformation under physiological conditions – protein’s primary structure dictates its three dimensional structure.
  • 89.
  • 90.
    Thermodynamics of ProteinFolding Lehninger 2000 Figure 6.27 Number of possible conformations is large and therefore the conformation energy is large. At this point only a small percentage of the intramolecular interactions found in the native state are present Decreasing number of possible states, i.e. the entropy Amount of protein in native state increasing Free energy is decreasing as we head to a free energy minimum at the bottom, i.e., native state. Energy max Energy min native min
  • 91.
    Protein Misfolding: Prions & Disease Lehninger 2000Box 6.4 •Brain becomes riddled with holes •Caused by a single 28 kd protein called a prion protein •Normal Prp is mostly alpha helical. •Illness occurs when an altered form of Prp called PrpScr is present which has mixed alpha helix and beta sheets Prion: proteinaceous infectious particle
  • 92.
    Alzheimers and -AmyloidProtein -Amyloid protein is normally present in the human brain but its function is unknown In Alzheimers a 40 residue (shorter) segment of this protein forms fibrous deposits or plaques. This cleaved protein tends to aggregate only with itself. This protein does not fold properly! Amyloid plaques w/ Aβ protein in brain tissue of human Alzheimers patient Native (N) <=> Unfolded (U) Keq = [U]/[N] = e-∆G˚’/RT As the ∆G˚’ for unfolding decreases the portion unfolded proteins increases.
  • 93.
    Proteins that HelpNative Protein Folding Molecular chaperones – proteins that bind to unfolded and partially folded polypeptide chains to prevent improper folding by prevent improper association of hydrophobic regions that could lead to polypeptide aggregation, precipitation, or non-native folding. Important for multisubunit and multidomain proteins. First described as heat shock proteins (Hsp) Most are ATPases Voet, Voet & Pratt 2012 Figure 6.45
  • 94.
    Proteins are DynamicStructures Myoglobin Voet, Voet & Pratt 2013 Figure 6.39 •Remember that proteins are in fact dynamic molecules with normal structural fluctuations •These fluctuations are important for function particularly in enzymes. •Snapshots are for the structure of myoglobin seen over 4 x 10-12 seconds
  • 95.