2GoalsUnderstand 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.
3Levels of Protein Structure PrimarySecondaryTertiaryQuaternaryRefer to the three-dimensional shape of folded polypeptide chains
4Protein DiversityFor a protein of n residues there are 20n possible sequencesFor 40 residue protein x 1052For 100 residue protein x 10130DefinitionsPeptides – typically less than 40 residuesDipeptide: 2 amino acids; Tripeptide: 3 amino acidsOligopeptide: many amino acidsProteins – typically polypeptides with 40 or more residuesMultisubunit proteins - proteins with several identical or nonidentical subunits.
5PRIMARY STRUCTURE & ANALYSIS Primary Structure: the amino acid sequence of a protein’s polypeptide chain or chains. Sometimes referred to as the covalent structure.
6Conjecture 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.
7Logic 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
10Protein PurificationCrude 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..
11Proteins 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
12Assay 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 helpfulCan also use a coupled enzyme reaction, i.e. 2nd enzymeCan use immunochemical assays.
14Some Separation Techniques Charge Ion Exchange ChromatographyElectrophoresisPolarity Hydrophobic Interaction ChromatographySize Gel Filtration ChromatographySDS-Polyacrylamide ElectrophoresisUltracentrifugationBinding Specificity Affinity ChromatographyThese separation techniques utilize differences in the physical and/or chemical properties that arise from the differences amino acid composition.
15Protein Fractionation by Salting Out Protein solubility depends on:Concentration of dissolved saltsSolvent polaritypHTemperatureBy careful manipulation of these properties it is possible to selectively precipitate out certain proteins and leave the other soluble.Voet, Voet & Pratt 2013 Figure 5.5
16Protein Separation by Ion Exchange Chromatography Ion exchange chromatography makes use of the fact that opposite charges attractPolyelectrolytes such as proteins that have both negative and positive charges will bind to cation or anion exchangers depending on the protein’s net chargeThe 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-Voet, Voet & Pratt 2013 Figure 5.6
17Protein Separation by Gel Filtration Chromatography 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.Voet, Voet & Pratt 2013 Figure 5.7
18Protein Separation by Affinity Chromatography 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 phaseColumn conditions are then changed, e.g. pH, ionic strength or high ligand concentration, to permit the protein to elute in a highly purified form.Voet, Voet & Pratt 2013 Figure 5.8
19SDS-PAGE of Supernatants & Membrane Fraction from a Bacterium Gel electrophoresis - a molecular sieving approachSDS, sodium dodecyl sulfate, when added to a protein solution binds 1 molecule of SDS per two amino acids or 1.4g per g protein.Voet, Voet & Pratt Figure 5.9 & 2013 Fig 5.9 & 5.10
21Protein SequencingA 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.Sequence is a prerequisite for determining protein’s 3-D structure and understanding its molecular mechanism.Sequence comparisons among analogous proteins from different species yield insights into protein function as well as reveal evolutionary relationships among proteinsMany 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
22Sanger Method for Protein Sequencing Bind to terminal amino groups to form a yellow dinitrophenyl derivativeHydrolyze proteinIdentify terminal amino acid chromatographicallyAlso today nucleic acids sequencing is frequently used to determine protein sequencesVoet, Voet & Pratt p.108 Box 5.1
23Basic Logic of Protein Sequencing 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 sequence5. Repeat process without cleaving the disulfide bonds to determine where those bonds are locatedVoet, Voet & Pratt 2013 Figure 5.13
24End Group AnalysisUsed 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.
25Protein Sequencing End Group Analysis with Danzyl Chloride Conjugate the fluorophore to primary amine(s).Perform an acid hydrolysisIdentify via chromatography the labeled amino acid.Voet, Voet & Pratt 2013 Figure 5.14
26Disulfide Bond Cleavage Need to cleave to separate polypeptide chains.Two methods:oxidative cleavage with performic aciddisadvantage: destroys met and Trp indol side chainreductive cleavage with mercaptane.g. 2-mercaptoethanol. Usually alkylate product with iodoacetic acid to prevent disulfide bond reformation.
27Peptide Hydrolysis Chemical approach Acid hydrolysis disadvantages – destroys Ser, Thr, Tyr & Trpconverts Asn & Gln to Asp & Glu, respectivelyBase Hydrolysisdisadvantages – destroys Cys, Ser, Thr & ArgBiochemical ApproachEnzymatic Hydrolysisdisadvantages – often incomplete some autodigestion
28Amino Acid Analysis Separation by HPLC Complete hydrolysis will yield the composition but not the sequenceVoet, Voet & Pratt 2006 Figure 5.15
29Molecular Mass Determination by Mass Spectrometry Electrospray ionization mass spectrometryPeptide in solution sprayed from capillary tube at high voltage to produce highly charged dropletsSolvent soon evaporates to give peptide ions in gas phase– Yield to +2.0 charge per kilodalton from, e.g., Arg & Lys protonationMeasures mass/charge (m/z) ratioElectrospray ionization (ESI) does NOT destroy proteins as earlier mass spec techniques did.Matrix-assisted Laser desorption/ionization –time of flight MALDI-TOFPermits 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.ESI mass spectrum of horse heart apomyolobinMass 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+ ionCan take two adjacent peaks and solve two linear equations to get MW.Berg, Tymoczko, & Stryer 2012 Fig 3.34Voet, Voet, & Pratt 2013 Fig 17a,b
30Tandem Mass Spectroscopy for Peptide Sequencing Tandem refers to two mass spectrometers in seriesIons 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 producedEach 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”Berg, Tymoczko, & Stryer 2012 Fig 3.36Voet, Voet & Pratt 2013, Fig. 5-18; & p. 113
31Polypeptide 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≠ProThermolysin Rn = Ile, Met, Phe, Try, Val Rn≠Pro (N-Side)Pepsin Rn = Leu, Phe, Trp, Typ; Rn≠Pro (N-side)Endopeptidase V8 Rn-1 = GluCyanogen Bromide (CNbr) Rn = Met (C side)Voet, Voet, & Pratt 2013 Table 5.4Endopeptidases hydrolyze internal peptide bonds and are used to fragment polypeptides but require certain adjacent side chains.
32Edman Degradation of Proteins 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.Voet, Voet & Pratt Figure 5.16
37Cytochrome c Phylogenetic Tree Voet, Voet & Pratt Figure 5.22
38Protein Evolution, Gene Duplication & Protein Modules Protein evolution ratesThe 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 functionGene duplicationProteins 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 modulesNew proteins (and functions) can also be generated by incorporation of various amino acid module or motifs.
39Sample Protein Evolution Rates Proteins mutate at different rates over time.But mutations in the DNA typically occur at the same rateDifferences due to the rate at which functionally or structurally acceptable changes occurs. That is those changes that are NONLETHALVoet, Voet & Pratt 2013 Figure 5.24
40SECONDARY STRUCTUREThe local spatial arrangement of a polypeptide’s backbone atoms without regard to the conformation of its side chains.
41Levels of Protein Structure Voet, Voet & Pratt 2013 Figure 6.1
42The (trans) Peptide Bond - Structure In most cases in the protein backbone the peptide bond is in the trans configurationMeans -carbons of adjacent amino acids are on opposite sidesSo less steric hindrance of adjacent amino acids side chainsFind ~ 8 kJ greater stability of the trans vs the cis configurationBond angles and lengths effect to a large extent the freedom of movement and the configuration of the protein. (Important!)Resonance give rise to 40% double bond characterVoet, Voet & Pratt Figure 6.2
43Polypeptide: Extended Conformation & Torsion Angles “peptide group”Definition: and = 180º when the polypeptide chain is fully extended. They increase clockwise when looking from CαVoet, Voet & Pratt 2013 Figure 6.3; 6.4
45Ramachandran Diagram (Allowed Bond Angles ) ↑↑ -pleated sheetα-helix (left handed)Sterically allowed angles for all aa except Gly & Provan der Waals radii, the attractive and repulsive forces we covered in earlier lectures have a significance for protein structure..α-helixNote: Gly is less sterically hinderedVoet, Voet & Pratt 2013 Figure 6.6
46Protein α-Helix Structure Amide HThe alpha helix structures is one of the most stable and is therefore one of the most abundant biological structures.H-bond every 4th residueCarbonyl OThe 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.5.4ÅRight-handed helixHelix core is tightly packed such that the atoms are at or near their van der Waals radii.leftrightVoet, Voet & Pratt 2013 Figure 6.7Lehninger Box 6.1
47-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-bondsMany adjacent Lys and/or Arg residues with pos. charges will repel each other at pH 7.0The bulk & shape of Asn, Ser, Thr and Leu can also destabilize a helix if close together in the backbone sequenceThe 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 formationAromatic 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
48Constraints on -Helix Stability (Summary) Electrostatic repulsion or attraction between successive amino acids with charged R groups.Bulkiness of adjacent R groups.Interactions between amino acid side chains spaced 3 (or 4) residues apart.The occurrence of Pro or Gly residues.Interaction between amino acid residues at the ends of the helical segment and the inherent electric dipole of the helix.
49Electric Dipole of the Peptide Bond & Interactions Between -Helix Residues Three Apart Arg103 side chainAsp100 side chainaminocarbonylThe 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.Troponin c protein segmentLehninger Figure 6.6Lehninger Figure 6.5
50Protein -Sheet Structures -sheet makes full use of the hydrogen bonding capacity of the polypeptide backboneH-bonding occurs between neighboring polypeptide chains, i.e. interchain, rather than intrachain.-Sheets: parallel vs AntiparallelSpace-filling Antiparallel-SheetPleated -SheetVoet, Voet & Pratt 2013 Figures 6.9, 6.10, 6.11
51Historical 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
52Fibrous ProteinsFibrous 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.
53-Keratin Structure: coiled coil Rich in Ala, Val, Leu, Ile, Met and Phe – hydrophobic residuesA “Permanent Wave”A coiled coil - composed of two parallel -helices that are twisted around each other to form a left-handed supertwisted coiled coil.Voet, Voet & Pratt 2013 Figure 6.15Lehninger Box 6.2
54Silk fibroin -Sheets in Side View Has great strengthNot very extensible (would break polypeptide chain covalent bonds)Very flexible (Neighboring sheets associate with weak van der Waals forces).Ala or SerGlyTypical repeat:(Gly-Ser-Gly-Ala-Gly-Ala)nVoet, Voet & Pratt 2002 Figure 6.16
55Collagen Triple Helix Structure Composition:~33% Gly~15-30% Pro, Hyp and Hylmost abundant vertebrate protein occurringfibers form the major stress bearing components of connective tissuesThree parallel, left-handed helical polypeptide chains with three residues per turn twisted together to form a right-handed superhelical structure.Repeating Sequence:Gly-X-Y where:X is often Pro, Y is often Hyp. Hyl is sometimes at YHyp = 4-hydroxyprolylHyl = 5-hydroxylysylVoet, Voet & Pratt 2013 Figure 6.17
56Collagen’s Molecular Interactions Every third polypeptide residue passes through the very crowded center of the superhelix, hence the repeated gly every third residue.H-BondSpace-filling modelBall & stick modelH-bonding in collagen triple helixVoet, Voet & Pratt 2002 Figure 6.18Voet, Voet & Pratt 2013 Figure 6.18a
58Nonrepetitive 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 descriptionGlobular 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.
59Turn & Loop Structures in Polypeptides Reverse turn types Loop in space-filling modelfound in most proteins with 60 are more residues and are composed on 6 to 16 residues.Voet, Voet & Pratt 2013 Figure 6.14Voet, Voet & Pratt 2006 Figure 6.20 Loop almost always located on the protein surface. May be involved in recognition processes.
60Structure of -turns hydrogen oxygen 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 acidsMost common type of turnLehninger Figure 4.8
61Relative Probability of an AA Being in These Secondary Structures Take home message: chemical and physical characteristics of an amino acid (charge, bond angles, etc.) influence its ability to participate in particular secondary structures(for illustrative, informational purposes only)Lehninger Figure 4.10
62TERTIARY STRUCTUREThe three-dimensional structure of an entire polypeptide including its side chainsTertiary structure describes the folding of the protein’s secondary structure elements and also specifies the position of each atom in the protein.
63Myoglobin Tertiary Structure: View Types ribbonmeshSurface contourMyoglobin is composed of eight relatively straight alpha helices interrupted by bends and some of these are beta turns.Space-filling w/ side chainsRibbon w/ side chainsLehninger Figure 4.16
64Protein 3-D structure & X-ray crystallography Protein crystal: flavodoxin from Desulfovbrio vulgarisX-ray diffraction pattern of sperm whale myoglobin crystal3-D electron density of human rhino virus crystalVoet, Voet & Pratt 2013 Figure 6.21Voet, Voet & Pratt 2002 Figure 6.23Voet, Voet & Pratt 2013 Figure 6.20b
653-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 structureLehninger Box 4.4
66Myoglobin, Globular Proteins, & Tertiary Structure Positioning of amino acid side chains reflects a structure that derives much of its stability from hydrophobic interactionsA 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.
67NMR in Protein Structure Determination Only certain atoms such as 1H, 13C, 15N, 19F, and 31P give rise to an NMR signal.1-DNMR used to identify nuclei and their immediate chemical environment. Also use NOE signals provide information about the distance between atoms#12-DLehninger Box 4.4 Fig 2
68NMR in Protein Structure Determination of Full Structure from 2D Spectrum Backbone showing possible constraintsPart 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 polypeptide20 possible structures shown w/ backbone in whiteVoet, Voet & Pratt Fig 6.25
69Protein 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.Val, Leu, Ile, Met & Phe occur mostly in the protein interior away from aqueous solvent molecules.Arg, His, Lys, Asp, & Glu are typically located at the proteins surface where their charges can be solvatedSer, 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.
70Side Chain Locations Seen in Space-filling Models sperm whale myoglobinconcanavalin A-sheet interior this sideNonpolar side chainsPolar side chainsbackbone-Sheetα-HelixVoet, Voet & Pratt 2008 Figure 6.26
71Horse Heart Cytochrome c Structure Fe atom & hemeHydrophillic side chainsHydrophobic side chainsHydrophilic sides chains are shown in green and can be seen to be at the protein’s surface.The hydrophobic side chains are in orange and are closer to the protein’s interior and near the porphyrin ring.Voet, Voet & Pratt 2013 Figure 6.27
72Protein 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. motifAntiparallel strands connected by relatively tight reverse turns hairpinTwo 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
73Protein Structural Motifs Different proteins combine these structures in various ways to achieve their function. Certain successful and energetically favorable designs are preserved in many diverse proteins.α hairpinα α barrelsVoet, Voet & Pratt 2008 Figure 6.28
74The Rossman Fold Nucleotide binding site A prime example of a structure function-relationshipBinds dinucleotides such as NAD+.Utilizes strands which form a parallel sheet with -helical connectionsTwo such units are shown.Voet, Voet & Pratt 2013 Figure 6.31Voet, Voet & Pratt 2002 Figure 6.29
75Protein DomainsPolypeptide 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 proteinDomains 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.
76Domains in EvolutionCommon protein structures likely arose and persisted because of their ability to:Form stable folding patterns.Tolerate amino acid deletions, substitutions, & insertions which makes them better able to survive evolutionary changes.Support essential biological functions.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.
77QUATERNARY STRUCTUREThe spatial arrangement of the subunits of a multisubunit proteinSubunits typically associate via noncovalent interactions.Contact regions between subunits resemble the interior of single subunit proteins.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.Definitions: Oligomer – proteins with more than one subunitProtomer – repeating structural subunits of a protein
78Quaternary Structure of Hemoglobin An oligomeric proteinEach of the four subunits 1212 is shown in a different color. (Heme is red)Voet, Voet & Pratt 2008 Figure 6.33
79Oligomeric Protein Symmetry Examples Related by single axis of rotationProteins can only have rotational symmetryWhen n-fold rotation axis intersects a 2-fold rotation axis at 90°Other types of symmetry based on geometrical objectsVoet, Voet & Pratt 2013 Figure 6.34
80Protein 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 unfoldedConclude: a protein’s structure is in fact a delicate balance of counteracting forces.
81Stabilizing ForcesHydrophobic 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
82Hydropathy Plot Protein interior 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.Protein exteriorVoet, Voet & Pratt 2008 Figure 6.35
83Electrostatic 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.
84Chemical Cross-Linking Disulfide BondsThought 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 Ionscan also internally link proteins
85Electrostatic 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.
86Protein 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 interactionsChaotropic agents (urea, guanidinium chloride) Ions or small organic molecules that at high 5- 10M concentration disrupt hydrophobic interactions
88Protein RenaturationAnfinsen 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.
90Thermodynamics of Protein Folding 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 presentnative minEnergy maxDecreasing number of possible states, i.e. the entropyAmount of protein in native state increasingFree energy is decreasing as we head to a free energy minimum at the bottom, i.e., native state.Energy minLehninger Figure 6.27
91Protein Misfolding: Prions & Disease Brain becomes riddled with holesCaused by a single 28 kd protein called a prion proteinNormal Prp is mostly alpha helical.Illness occurs when an altered form of Prp called PrpScr is present which has mixed alpha helix and beta sheetsProtein Misfolding: Prions & DiseasePrion: proteinaceous infectious particleLehninger Box 6.4
92Alzheimers and -Amyloid Protein -Amyloid protein is normally present in the human brain but its function is unknownIn 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 patientNative (N) <=> Unfolded (U)Keq = [U]/[N] = e-∆G˚’/RTAs the ∆G˚’ for unfolding decreases the portion unfolded proteins increases.
93Proteins 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 ATPasesVoet, Voet & Pratt 2012 Figure 6.45
94Proteins are Dynamic Structures Myoglobin Remember that proteins are in fact dynamic molecules with normal structural fluctuationsThese fluctuations are important for function particularly in enzymes.Snapshots are for the structure of myoglobin seen over 4 x secondsVoet, Voet & Pratt 2013 Figure 6.39