Amino acids-proteins• I. Overview• Most diverse and abundant molecules in living systems• Functional components: enzymes, hormones, cell- surface receptors• Structural components: cell membranes, organelles; bone, skin, muscle, connective tissue• Other specialized roles: immunoglobulins, hemoglobin, albumin
II. Structure of Amino Acids• More than 300 amino acids known, but only 20 coded for by DNA• At pH 7.4 (physiological pH), amino acids exist in zwitterionic form (positive NH3+ and negative COO- charges).• Classified based on side chain (R) group: Nonpolar, Polar, Charged (acidic or basic)
A. Amino acids with non-polar side chains• do not bind nor give protons• do not form hydrogen bonds• have hydrophobic interactions• 1. Location of non-polar (hydrophobic) amino acids in proteins – In soluble proteins (aqueous environment), found in the interior of proteins (shielded from environment) – In membranes or other hydrophobic environments, found on protein surface. – Proline: side chain forms an imino group
B. Amino acids with uncharged polar side chains
• 0 charge at neutral pH• Cys & Tyr can lose a proton at alkaline pH• Ser, Thr & Tyr – polar –OH can form hydrogen bonds• Asn & Gln contain –COOH (carboxy) and – CONH2 (carboxyamine) groups – can form hydrogen bonds.
• 1. Disulfide bond:• Side chain of Cys contains –SH group – important active site of enzymes• Proteins with 2 –SH groups can form a disulphide bridge or cystine dimer (-S-S- , intermolecular or intramolecular).
B. Amino acids with uncharged polar side chains• 2. Side chains as sites of attachments for other compounds:• Ser, Thr & Tyr contain polar –OH group – site of attachment for PO4- group, for e.g. Ser side- chain important active site component in many enzymes – -CONH2 group of Asn and –OH group of Ser & Thr serve as site of attachment of oligosaccharide chains in glycoproteins
C. Amino acids with acidic side chains• Asp & Glu are proton donors.• At neutral pH (physiological), side chains fully ionized or dissociated (COO-) and carry a net negative charge.• Contribute a negative charge to proteins .• Aspartate (aspartic acid) and glutamate (glutamic acid).• R groups typically have a pK< 7
D. Amino acids with basic side chains• Side chains of basic amino acids accept protons• At physiologic pH, side chains of Lys and Arg are fully ionized – positively charged ( NH3+)• Contribute a positive charge to proteins that contain them• Have a pK value>7( histones have an abundance of Arg and lys, net +ve charge)• His -- weakly basic and partially positively charged at physiologic pH- good buffering capacity• In proteins, can be +ve or –ve depending on environment of protein (important role in proteins like myoglobin).
E. Abbreviations and symbols for commonly occurring amino acids3-letter abbreviation and one-letter symbol1. Unique first letter Cys CCysteineHistidine His HIsoleucine Ile IMethionine Met MSerine Ser SValine Val V
2. Most commonly occurring amino acids have priority Ala AAlanineGlycine Gly GLeucine Leu LProline Pro PThreonine Thr T
3. Similar sounding names Arg R (“aRginine)ArginineAsparagine Asn N (contains N)Aspartate Asp D (“asparDic”)Glutamate Glu E (“glutEmate”)Glutamine Gln Q (“Q-tamine”)Phenylalani Phe F (“Fenylalanine”)neTyrosine Tyr Y (“tYrosine”)Tryptophan Trp W (double ring in the molecule)
4. Letter close to initial letter: Asx B Aspartate or asparagines Glutamate or Glx Z glutamine Lysine Lys K (near L) Undetermined X amino acid
F. Optical properties of amino acids:• α-C of each amino acid attached to 4 different chemical groups• α-C is chiral or optically active i.e. it has four different groups attached to the -carbon (except Gly). The number of optical isomers is 2n, where n is the number of chiral atoms in the molecule.• 2 stereoisomers, optical isomers or enantiomers: D- and L- forms are mirror images of one another, only L-forms found in human bodies
I. Overview• 20 amino acids linked together with peptide bonds• 4 organizational levels: primary, secondary, tertiary and quaternary
• Primary Structure of Proteins• Sequence of amino acids = primary structure• Genetic diseases result from proteins with abnormal sequences
Primary structure: insulin
Peptide Bond• Not broken when proteins are denatured• Prolonged exposure to acid or base at high temps is necessary to break bonds.
• 1. Naming the peptide• a. order of amino acids in a peptide• Left (N-terminal a.a.) is written first, C-terminal next• b. Naming of polypeptides• component a.a. in peptides called moieties or residues.• Except C-terminal, all moieties called –yl instead of – ine –ate, or -ic• E.g. valylglycylleucine
Characteristics of the peptide bond:• a. Lack of rotation around the bond:• partial double bond- rigid and planar. bond between -C and -amino or –CO group is rotatable• b. Trans configuration:• (steric interference in cis position)• c. Uncharged but polar:• like all –CONH2 links, peptide bonds do not protonate between pH 2-12• only side chains and N- and C- terminals can ionize• peptide bond is polar (uncharged) and can be involved in H-bonding.
Characteristics of the peptide bond 3. Trans configuration • minimizes steric hindrance
A peptide bond is formed from a condensation reaction (dehydration) involving twoamino acids.A molecule of H2O is eliminated.
Dipeptide formation H H H O O N C C H H3N C C O H CH O CH3 H3C CH3 alanine valine H2O peptide bond H O H O amino carboxyl H3N C C N C C terminus terminus H O ( amino group) CH3 CH H3C CH3 alaninylvaline
Characteristics of the peptide bond H O H OH3N C C N C C H O R1 R2
Characteristics of the peptide bond H O H O 1. partial double-bond character H3N C C N C C • due to resonance H O R1 R2 O R2 O R2 H H C C O C C O C N C C C NH3N H H3N H H O H O R1 R1 O R2 H C C O C C N H3N H H O R1
Characteristics of the peptide bond 2. rigid and planar • rotation occurs around single bonds but not around double bonds no rotation around peptide bond H O H O H3N C C N C C H O R1 R2
O O H CRotation around single bonds H C C O C N R2 H3N H R1Because no rotation is possible around O R2double bonds, the stereochemistry of the Hpeptide bond is fixed. C C O C N C H3N H H O R1 O R2 R1 C C O H C N C H H O NH3
Characteristics of the peptide bond 4. Uncharged but polar • dipole moment exists due to separation of charge O R2 H C C O C C N H3N H H O R1
Characteristics of the peptide bond - summary • partial double bond character • rigid and planar • trans configuration • uncharged but polar amide trans plane config O R2 H C C O C N C H3N H H O R1
B. Determination of the amino acid composition of a polypeptide• First, identify and quantify constituent amino acids.• Pure sample must be used, contamination gives errors.• 1. Acid hydrolysis:• Hydrolyzed by strong acid at 110 C for 24 h• Peptide bonds cleaved• Gln & Asn Glu & Asp; Trp mostly destroyed• Procedure gives composition but not sequence
• 2. Chromatography:• Individual aa’s separated by cation-exchange chromatography• Anion-exchange resin for -vely charged aa’s• Eluted from column by buffers of increasing ionic strength and pH• aa’s separated at different ionic strength and pH• 3. Quantitative analysis:• Quantified with ninhydrin purple compd. with amino acids, NH3 and amines (yellow color with imino group of Pro).• Intensity of color measured in spectrophotometer• Area under curve proportional to amount of amino acid• If MW of protein known, no. of residues of each aa known, otherwise, only ratio of no. of molecules of each amino acid determined.• Done using amino acid analyzer
C. Sequencing of the peptide from its N-terminal end • Phenylisothiocyanate – Edman’s reagent – used to label N-terminal res under mildly alkaline conditions. phenylthiohydantoin (PTH). • This makes N-terminal residue peptide bond weak; break it without breaking others. • Above process occurs in a cycle to sequence peptide using “sequenator” • Can be used for polypeptides of 100 a.a. or less.
OH2N CH C Lys His Phe Leu Arg COOH CH3 N C SN-terminal 1. Labeling Phenylisothiocyanate alanine (Edman’s reagent) H O HN CH C Lys His Phe Leu Arg COOHS C CH3 labeled peptideH N 2. Acid cyclization and expulsion of hydrolysis shortened peptide chain O S + CH2N CH C His Phe Leu Arg COOH N NH (CH2)4 C CH O NH 2 CH3 N-terminal PTH-alanine lysine
Cleavage of peptide into smaller fragments • occurs before Edman degradation • necessary if peptide is > 100 amino acids in length • need to use more than one cleaving agent in order to determine amino acid sequence • different enzyme/chemical specificity • overlap peptide fragments in order to determine original sequence
• 2. Chemical Cleavage:• Cyanogen bromide cleaves polypeptides on –CO side of methionine residue• 3. Overlapping peptides:• Individual peptides sequenced by Edman’s degradation• Overlapping peptides help determine sequence• 4. Multimeric proteins:• Multiple peptides separated (H-bonds and noncovalent bonds) by urea or guanidine.HCl• Disulfide bridges broken with performic acid.
• Secondary structures result from localarrangement of adjacent amino acids into anorganized 3- dimensional structure.• H-bonds are key to stabilizing these structures.Secondary structures include:• Helical Structures• Beta Structure (maximally extended primary sequence)• Random chain (nonrepetitive)
HelixLeft-hand Right-hand helix helix
Intrachain Hydrogen Bonding is important in maintaining secondary protein structure.Here (in the α helix) the carbonyl oxygen from one amino acid is H-bonded to an alphanitrogen of the 4th distant amino acid in the polymer. Hydrogen bond
• 3.6 residues per turn• R groups extend outward helix is disrupted by:1) P and G2) large numbers of charged aa’s3) aa’s with bulky R groups
Sheet• “pleated”• all peptide bond components involved in H-bonding• strands visualized as broad arrows N terminal C terminal• may be parallel or antiparallel
-Bend• function to reverse the direction of polypeptide chain• often include charged residues• stabilized by ionic and/or H-bonds• usually composed of 4 amino acids including Pro and Gly
Supersecondary structure (motif)• result from local folding of secondary structures into small, discrete, commonly-observed aggregates of secondary structures:• loop• corner
• extended super secondary structures are known as domains• barrel• twisted sheet
• Tertiary structure is the 3 dimensional form of a molecule resulting from distant protein-protein interactions within the same polypeptide chain (caused by folding of secondary structures): Globular proteins are characterized as generally having: • a variety of different kinds of secondary structure • spherical shape • good water solubility • a catalytic/regulatory/transport role i.e. a dynamic metabolic function
• IV. Tertiary Structure of Globular Proteins• Tertiary structure – folding of domains and final arrangement of domains in protein• Compact, hydrophobic side chains buried in interior• Maximum hydrogen bonding of hydrophilic groups within molecule
Fibrous proteins are characterized as generallyhaving:• one dominating kind of secondary structure (i.e. collagen helix in collagen)• a long narrow rod-like structure• low water solubility• a role in determining tissue/cellular structure and function (e.g. collagen, keratin)
• Domains• Fundamental functional and 3-D structural units of polypeptides• >200 amino acids 2 or more domains• folding within domain independent of folding within other domains• each domain has characteristics of small, compact, globular protein
• 1. Disulfide bonds:• formed from –SH groups of two cysteine residues Cystine• two Cys may be close by or far away• stabilize the protein found in many secreted proteins• 2. Hydrophobic interactions:• interactions between nonpolar side chains of amino acids in interior of protein
• 3. Hydrogen bonds:• interactions between polar side chains• interactions between polar side chains and water enhanced solubility• 4. Ionic interactions:• e.g. Interaction of –COO- of Asp with NH3+ of Lys
Protein folding• Trial and error process that depends on• Composition of side chains• H-bonding• Disulfide bonds• Ionic interactions• To result in most stable or favorable structure• Chaperones: play a role in folding of proteins during their synthesis (separate, enhance the rate, protect residues).
Denaturation of Proteins• Destruction of all but primary structure• Denaturing agents: heat, organic solvents, mechanical shearing, heavy metals, detergents, chaotropic agents• May be reversible or irreversible• Loss of biological activity
Most proteins do not revert to their original tertiarystructures after denaturation.Ribonuclease enzyme is an exception.
Protein misfolding• Spontaneous• Mutation• Proteolytic cleavage, e.g. accumulation of amyloid plaques (amyloid-β) in Alzheimer’s.• Abnomal form of tau accumulation in neufibillary tangles of Alzheimer’s brain• Prion disease: Creutzfeldt-Jakob disease – humans• A protein- as a degenerative agent
-Sheet in fibrous (Amyloid) protein• Amyloid protein deposited in brains of Alzheimer’s disease patients – twisted -pleated sheet fibrils with 3-D structure virtually identical to silk fibrils
Quaternary structure consists of the associationof multimeric proteins (identical or nonidentical)held together by one or more of the followingnoncovalent interactions: Hydrogen bonds Hydrophobic interactions (Van der Waals forces) Electrostatic interactions (ionic and/or polar)