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1、 6 3D Structure of Proteins6.1 Overview of Protein Structure6.2 Protein Secondary Structure6.3 Tertiary & Quaternary Structure6.4 Protein Denaturation & Foldingliguofu6.1 Overview of Protein Structure6.1.1 A proteins conformation is stabilized largely by weak interactions6.1.2 The peptides bond is r
2、igid and planar liguofuIts not quaternary structureliguofu 6.1.1 A proteins conformation is stabilized largely by weak interactionsConformation is the spatial arrangement of all atoms in a protein. The possible conformations of a protein include any structural state that can be achieved without brea
3、king covalent. Because free rotation is possible around many single bonds, the conformations of a protein can be numberless.Native proteins are the proteins in any of their functional and folded conformations.This is usually the conformation that is thermodynamically the most stable, having the lowe
4、st free energy (G). The conformations of a native protein may be the lowest G state or at least near the lowest G state.liguofuStability can be definded as the tendency to maintain a native conformation. Native proteins are only marginally stable. Weak interactions:Hydrogen bondStatic electrical for
5、cevan der Waals forceHydrophobic interaction 6.1.1 A proteins conformation is stabilized largely by weak interactionsliguofuUnfolded proteinFolded proteinG = H-T S|G|=2065 kJ/mol生理条件下生理条件下Hydrogen bonds between groups in proteinsStatic electrical force between charged groups attraction or repulsionC
6、harged groups interact with ions in solutionGroups hydrogen-bonding with waterHydrophobic effect between hydrophobic groups and waterHigh conformational entropyLow conformational entropy Hydrophobic groups interactionDecreasing the entropy of waterIncreasing the entropy of water 6.1.1 A proteins con
7、formation is stabilized largely by weak interactionsliguofuConclusion:The decrease of the free energy during protein folding is largely from the increase of the entropy of water In other words, The net force is mainly the hydrophobic interactions that impulse a unfolded protein into a folded protein
8、. Hydrophobic residues are largely buried in the protein interior, away from water. Although the other weak interactions are not the impulsive force in the process of protein folding, they are important in maintaining the folded state of a protein. The number of hydrogen bonds within a protein inten
9、ds to be maximized. 6.1.1 A proteins conformation is stabilized largely by weak interactionsliguofuX-ray diffraction studies of AAs and of simple dipeptides and tripeptides revealed that the peptide (amide) bond is about 1.32 (C-N single bond, 1.49; C=N double bond, 1.27), thus having partial double
10、 bond feature (should be rigid and unable to rotate freely).The partial double bond feature is a result of partial sharing (resonance) of electrons between the carbonyl oxygen and amide nitrogen.The atoms attached to the peptide bond are coplanar with the oxygen and hydrogen atom in trans positions.
11、6.1.2 The peptides bond is rigid and planarliguofuLinus Paulingliguofu6.1.2 The peptides bond is rigid and planarliguofuThe backbone conformation of a peptide can be defined by two sets of rotation angles. The rotation angles around the N-C bonds are labeled as phi ( ), and around C -C bonds are psi
12、 ( ).By convention, both phi and psi are defined as 0 degree in the conformation when the two peptide planes connected to the same carbon are in the same plane.In principle, phi and psi can have any value between -180 and +180 degrees.The conformation of the main chain is completely defined when phi
13、 and psi are specified for each residue in the chain.6.1.2 The peptides bond is rigid and planarliguofuc) The conformation corresponding to =0o, =0o, which is disallowed by the steric overlap between H and O atoms of adjacent peptide planes.b) The conformation corresponding to =180o, =180o, when the
14、 peptide is in its fully extended conformation.6.1.2 The peptides bond is rigid and planarliguofuthe lightest blue area reflects conformations that are permissible if a little flexibility is allowed in the bond angles.Ramachandran plot for L-Ala residues.Dark blue area reflect conformations that inv
15、olve no steric overlap and thus are fully allowed; medium blue indicates conformations allowed at the extreme limits for unfavorable atomic contacts;liguofu6.2 Protein Secondary Structure6.2.1 -helix -chain6.2.2 -pleated sheet6.2.3 turnLoopRandom coilThe likely regular conformations of protein molec
16、ules were proposed before they were actually observed! This was accomplished by building precise molecular models.liguofuThe simplest arrangement of the polypeptide chain was proposed to be a helical structure called -helix (Pauling and Corey, 1951)The polypeptide backbone is tightly wound around th
17、e long axis (rodlike).R groups protrude outward from the helical backbone.A single turn of the helix (corresponding to the repeating unit in -keratin) extends about 5.4 Angstroms, including 3.6 residues (each residue arises 1.5 and rotate 100 degrees about the helix axis).6.2.1 -helixliguofuThe mode
18、l made optimal use of internal hydrogen bonding for structure stabilization.Each carbonyl oxygen of the residue n is hydrogen bonded to the NH group of residue (n+4).The residues forming one -helix must all be one type of stereoisomers (either L- or D-).L amino acids can be used to build either righ
19、t- or left-handed -helices (left-handed -helices have not been discovered in any organisms ).6.2.1 -helixliguofuliguofuliguofuliguofu& Five constraints affecting the stability of a-helixThe electrostatic repulsion (or attraction) between successive AA residues with charged R groups;The bulkiness of
20、adjacent R groups;The interactions between AA side chains spaced three (or four) residues apart;The occurrence of Pro and Gly residues;The interaction between AA residues at the ends of the helical segment and the electric dipole inherent to the helix.liguofu -pleated sheet (or conformation) was pro
21、posed to be the more extended conformation of the polypeptide chain.The conformation is formed when two or more almost fully extended polypeptide chains are brought together side by side.Regular hydrogen bonds are formed between the carbonyl oxygen and amide hydrogen between adjacent chains (look li
22、ke a zipper). 6.2.2 -pleated sheetliguofuThe axial distance between the adjacent amino acid residues is 3.5 Angstroms.The planes of the peptide bonds arrange as pleated sheets.The R groups of adjacent residues protrude in opposite directions.The adjacent polypeptide chains can be either parallel (th
23、e same direction) or antiparallel (the opposite direction). 6.2.2 -pleated sheetliguofuliguofuliguofu turn (hairpin发发卡卡 turn) is also a common secondary structure found where a polypeptide chain abruptly reverses its direction.It often connects the ends of two adjacent segments of an antiparallel -p
24、leated sheet.It is a tight turn of 180 degrees involving four amino acid residues.The essence of the structure is the hydrogen bonding between the C=O group of residue n and the NH group of the residue n+3. 6.2.3 turnliguofuGly and Pro are often found in turns. Gly is there (as the 3rd residue in ty
25、pe II) because it is small and flexible; for Pro it is because the peptide bond involving Pro can assume the cis configuration, which in turn generates a tight turn on the polypeptide chain. turns are often found near the surface of a protein. 6.2.3 turnliguofuWith different , anglesliguofuliguofuSo
26、me amino acid residues are accommodated in the different types of secondary structure better than others.The probability is calculated from known protein structures. It is used in predicting secondary structures.Some bias or propensities can be explained easily.Others are not yet understood.& AAs te
27、ndency in secondary structureliguofuRamachandran plots for a variety of structuresliguofuRelative probabilities that a given amino acid will occur in the three common types of secondary structure.liguofu6.3 Tertiary & Quaternary Structure6.3.3 Structrural patterns of globular proteins 6.3.1 Fibrous
28、proteins are adapted for a structural function6.3.2 Globular proteins are diversity in structure and function liguofu1. 1. -Keratin2.Collagen3.Silk fibroin 6.3.1 Fibrous proteins are adapted for a structural functionliguofu1. 1. -keratins -keratins are rich in hydrophobic residues, including Phe, Il
29、e, Val, Met, and Ala, that make the protein insoluble in water.Two helical strands ( -helix) wrap in parallel together to form a supertwisted coiled coil in -keratin.The superhelical twisting is left-handed in -keratins (opposite to the individual strand).The surfaces where two -helices touch are ma
30、de up of hydrophobic AA residues, their R groups meshed together in a regular interlocking pattern.liguofuliguofu -keratins are the main components of skin and many skin derivatives in vertebrate animals. Including, e.g., hair, wool, feathers, nails, claws, quills, scales, horns, hooves, tortoise sh
31、ell, and much of the outer layer of skin.Usually harder -keratins contain higher number of Cys (18% of the residues are Cys in tortoise shells and rhinoceros horns) involved in disulfide bonds . -keratins can be stretched (to twice as its original length) due to its structure springiness.1. 1. -kera
32、tinsliguofuWhile -keratins have relatively simple tertiary structure, their quaternary structure can be quite complex. -keratinsliguofu1. 1. -keratins: Permanent waving of hairliguofuCollagen has left-handed polypeptide chains (a unique secondary structure, three aa residues per turn, called - chain
33、) wrapped together to form right-handed triple helixThe amino acid sequence of collagen is revealed to be remarkable regular: a repeating tripeptide (Gly-X-Pro or Gly-X-Hyp, hydroxylproline, where the sequence Gly-Pro-Hyp recurs frequently). Only the small Gly can fit into the crowded interior of th
34、e triple helix, while Pro permits the sharp twisting of the collagen helix.2.CollagenliguofuliguofuIntrachain hydrogen bonds are absent, while interchain hydrogen bonds are formed.The rise per residue is 2.9 and there are nearly 3 residues per turn.Also superhelical twisting, but right handed, oppos
35、ite to that of a keratin.The collagen polypeptide chains within or between the triple helices are covalently cross linked through Lys or Hylys side chains.The superhelix provides great tensile strength with no capacity to stretch.Collagen fibers have similar tensile strength as a steel wire of equal
36、 cross section. chains versus helixliguofuliguofuliguofuCollagen is the most abundant protein in mammals. About 25% of the total protein mass in mammals is collagen.It is a major component of tendons, the extracellular matrix of the connective tissues (skin, bone matrix), and the cornea of the eye.C
37、ollagen triple helices (also called tropocollagen) self-assemble in the extracellular space to form much larger collagen fibrils that further aggregate into collagen fibers.2.CollagenliguofuThe collagen triple helices are regularly staggered in fibril to give rise to the striated appearance in negat
38、ively stained electron micrograph.The fibril formation involves many enzymatic steps. Deficiency of these steps generate many genetic diseases (e.g., osteogenesis imperfecta, Ehlers-Danlos syndrome, both resulted from single amino acid replacements of a Gly).2. Collagen: significanceliguofu3. Silk f
39、ibroinThe protein of silk is produced by insects and spiders.Fibroin is rich in Ala and Gly, permitting a close packing of sheets and an interlocking arrangements of R groups.Overall structure is stabilized by extensive hydrogen bonds and by optimization of van der Waals interactions between sheets.
40、Silk does not stretch since its conformation is already highly extended.Structure is flexible since the major force holding sheets together is weak interactions rather than disulfide bonds as in keratins.liguofuliguofuStrands of fibroin (blue) emerge from the spinnerets of a spider in this colorized
41、 electron micrograph.liguofu 6.3.2 Globular proteins are diversity in structure and function1.Myoglobin provided early clues about the complexity of globular protein structure2.Methods for determining the 3D structure of a proteinliguofuSperm whale myoglobin(肌肌红红蛋蛋白白), the oxygen carrier in muscle,
42、was the first protein to be seen in atomic detail by X-ray analysis (John Kendrew, 1950s, He and Max Perutz won the Nobel Prize in Chemistry in 1962 for determining the complete atomic structure of myoglobin and hemeglobin.). The existence of -helices were for the first time directly observed in a p
43、rotein. The myoglobin molecule contains eight -helices. All the-helices are right-handed. All the peptide bonds are in the planar trans configuration. There is no-pleated sheets observed in the molecule.1. MyoglobinliguofuliguofuThe myoglobin molecule has a dense hydrophobic core. Many hydrophobic R
44、 groups (e.g., Leu, Val, Met, Phe) are found to be in the interior of the myoglobin molecule.Only two hydrophilic histidine residues were found in the interior of the protein.All but two of the polar R groups are located on the outer surface and hydrated. (Nonpolar residues can be also present on th
45、e outer surface!)Bending are made of residues or sequences that are incompatible with -helical structure. All Pro residues are found at bends.1. MyoglobinliguofuThe flat heme group (the prosthetic group) was revealed to rest in a crevice (pocket). The heme group consists of a complex organic ring st
46、ructure, protoporphyrin, which is bound to an iron atom in its ferrous (Fe2+) state.The iron atom has six coordination bonds, with four in the plane of and bonded to the flat porphyrin molecule and two perpendicular to it.One of the perpendicular coordination bonds is bound to a nitrogen atom of an
47、interior His residue.The sixth coordination serves as the binding site for O2.The accessibility of the heme group to solvent is highly restricted, thus preventing the oxidation of the Fe2+ to the ferric ion (Fe3+), which is unable to bind O2.1. Myoglobinliguofu ribbon (only backbone)“mesh” imagesurf
48、ace contour imageribbonspace-filling modelliguofuliguofuThe heme groupliguofuGlobular proteins have a variety of tertiary structuresliguofuliguofu2. Methods for determining the 3D structure of a proteinFX-Ray diffraction crystallographyFNuclear magnetic resonance (NMR)FElectron crystallography (2D3D
49、)liguofuX-Ray diffraction crystallographyliguofuX-Ray diffraction crystallographyliguofuOne-dimensional NMRliguofuTwo-dimensional NMRliguofuTwo-dimensional NMRliguofuElectron crystallography2D3DReconstructionliguofuPrinciple for ReconstructionliguofuElectron crystallographyliguofuPick particles (man
50、ual or semiauto)Electron crystallographyliguofu1.Supersecondary structures and domains2.Protein motifs are the basis for protein structural classification3.Quaternary structures: Assembly & Size6.3.3 Structrural patterns of globular proteinsliguofuThe supersecondary structures, also called motifs or
51、 simply folds, refers to clusters of secondary structures that repeatedly appear.The already identified supersecondary structures include mainly motif, Greek key motif, -hairpin loop, four-helix-bundle, etc.Supersecondary structure motifs are usually also folding motifs of proteins. (a conjecture, N
52、ot completely established experimentally).A compact region (usually including less than 200400 residues) that is a distinct structural unit within a larger polypeptide chain is called a domain.Many domains fold independently into thermodynamically stable structures, and sometimes, have separate func
53、tions.1.Supersecondary structures and domainsliguofuStructural domains in the polypeptide troponin (肌钙肌钙蛋白蛋白) C, two separate calcium-binding domainsliguofuSupersecondary structureBurial of hydrophobic AA R groups requires at least two layers of secondary structures.Connection between elements of se
54、condary structure can not cross or form knots.The b conformation is most stable when the individual segments are twisted right-handed.Greek keyliguofuTwo particularly stable arrangements of adjacent chains: the barrel and the saddle; these structures form the stable core of many proteins liguofuRepe
55、ated usage of a patternConstructing large motifs from smaller ones. The / barrel is a common motif constructed from repetitions of the simpler - - loop motif. This / barrel is a domain of the enzyme pyruvate kinase from rabbit. liguofu2. Protein motifs are the basis for protein structural classifica
56、tionliguofuliguofuliguofuliguofu3.Quaternary structures: Assembly & SizeliguofuDeoxyhemoglobin 2 2Heme groupliguofuDeoxyhemoglobin 2 2Heme groupliguofuliguofuliguofuHelical symmetryrod-like or filamentous structuresliguofuCapsid of tobacco mosaic virus: the virus-encapsulating cylindrical structure
57、is a right-handed helical filament made up of 2,130 identical subunits.Size limit: the genetic coding capacity; the error frequency during protein biosynthesisliguofuliguofuIcosahedral SymmetrySolid with 20 triangular faces: There are, in fact, six 5-fold axes of symmetry passing through the vertice
58、s, ten 3-fold axes extending through each face and fifteen 2-fold axes passing through the edges of an icosahedron. Closed shell with the smallest number (60) of identical subunitsMore than 60 subunits-quasiequivalent positionliguofuIcosahedral SymmetryliguofuTriangulation numberDescription of the t
59、riangular face of a large icosahedral structure in terms of its smaller triangular faces (facets)liguofuNodamura VirusesliguofuPoliovirusliguofuHuman poliovirus (脊髓灰质炎病毒脊髓灰质炎病毒) has an icosahedral capsid (衣壳衣壳, coat protein of virus). liguofuAdenovirus liguofuSindbis Virusliguofu6.4 Protein Denatura
60、tion & Folding6.4.1 Loss of structure results in loss of function6.4.2 Denaturation of some proteins is reversible6.4.3 Polypeptides fold rapidly by stepwise process6.4.4 Death by misfolding: the prion diseases6.4.5 Protein folding in vivoliguofuProteins are relatively easy to lose their tertiary st
61、ructures due to their marginal stability maintained by noncovalent interactions.The process of total loss or randomization of three-dimensional structure of proteins is called denaturation.Protein denaturation results from a change in the solvent environment that is sufficiently large to upset the f
62、orces that keep the protein structure intact.Many means can cause protein to denature: heating; extreme pH; Miscible organic solvents (alcohol and acetone); solutes (urea, guanidine, provide alternative hydrogen bonding); detergents (SDS, introducing their hydrophobic tails into the proteins interio
63、r).6.4.1 Loss of structure results in loss of functionliguofuSome denatured globular proteins will regain their native structure and their biological activity once returned to conditions in which the native conformation is stable. This process is called renaturation.The denaturation and renaturation
64、 phenomena were originally observed on ribonuclease A by chance by Christian Anfinsen (1950s).Ribonuclease A became reduced and randomly coiled (denatured) in 8 M urea plus b-mercaptoethanol, with a loss of the enzymatic activity.6.4.2 Denaturation of some proteins is reversibleliguofuWhen urea and
65、-mercaptoethanol were removed, the enzymatic activity was slowly regained until full recovery under stable conditions, with existence of trace amount of -mercaptoethanol.All the physical and chemical properties of the refolded enzyme were virtually identical with those of the native enzyme.Conclusio
66、n: the information needed to specify the complex tertiary structure of ribonuclease A is all contained in its amino acid sequence.Subsequent studies have established the generality of this central principle of molecular biology: sequence specifies conformation.Nobel Prize in Chemistry in 1972 to Anf
67、insen.liguofuliguofue.g.liguofuliguofuliguofuThe tertiary structures of proteins are not rigid.Many studies have found that globular proteins have certain amount of flexibility in their backbones and undergo short-range internal fluctuations.Many proteins undergo small conformational changes in the
68、course of their biological function (e.g., O2-bound hemoglobin differs from O2-free hemoglobin, substrate binding to enzymes often causes conformational changes).liguofuThe protein folding problem is one of the most challenging and important areas of inquiry in biochemistry. How does the amino acid
69、sequence of a protein specify its three-dimensional structure? How does an unfolded polypeptide chain acquire the form of its native conformation?Are all possible conformations searched to find the energetically most favorable one? The Levinthals paradox: the huge difference between the calculated (
70、theoretical) time it may take for a polypeptide to fold by random searching and the actual time it takes. The cumulative selection, that is, partially correct intermediates are (recognized by nature and) retained due to sub-stability, makes the searching process much more efficient.6.4.3 Polypeptide
71、s fold rapidly by stepwise processliguofuliguofuProtein folding is an intriguing problem for both theoreticians and experimentalists. Proteins are only marginally stable. The free energy difference between the folded and unfolded states of a typical 100-residue protein is only 10 kcal/mol, meaning t
72、hat correct intermediates can be easily lost. The criterion of correctness is the total free energy of the transient species, not a residue-by-residue scrutiny of conformation. Some intermediates, called kinetic traps, have a favorable free energy but are not on the path to the final folded form.Mol
73、ten globules are formed early in folding. Molten globule state contains native secondary but not tertiary structure (an experimental observation). Hydrophobic collapse and acquisition of stable secondary structure are mutually reinforcing events in the formation of molten globules (synergistic, help
74、ing each other).liguofuPartially folded intermediates can be detected, trapped, and characterized. Rapid-kinetics studies, where protein secondary structures are monitored by spectroscopic methods (e.g., fluorescence, circular dichroism), can reveal the progression of distinctive intermediates durin
75、g refolding processes. Disulfide-bonded intermediates can be trapped covalently by blocking uncombined cysteines with iodoacetate. Pulsed hydrogen-deuterium exchange can be used to monitor the acquisition of secondary structures in protein folding. Our understanding of protein folding can be stringe
76、ntly tested by designing novel proteins with distinctive functions. For example, encouraging starts have been made in synthesizing new scaffolds, metal-binding proteins, channels, and catalysts.liguofuliguofuliguofu6.4.4 Death by misfolding: the prion diseasesA stained section of the cerebral of a p
77、atient with Creutzfeldt-Jakob diseaseBest known: Mad cow disease liguofuProtein folding in vivo is sometimes catalyzed by isomerases and chaperone proteins.The formation of correct disulfide pairing in nascent proteins is catalyzed by protein disulfide isomerase (PDI), which is especially important
78、for accelerating disulfide interchange in kinetically trapped folding intermediates.Peptidyl prolyl isomerases (PPIases) accelerate cis-trans isomerization of Pro residues during protein folding.6.4.5 Protein folding in vivoliguofuMolecular chaperones are proteins that interact with partially folded
79、 or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur (including folding, refolding, formation of oligomeric complexes, etc). Two classes of molecular chaperones have been well-studied.FHsp70 family: heat shock proteins of
80、 Mr70000, more abundant in heat stressed cellsFChaperonins: e.g. GroEL/GroES system (“Gro” refer to the necessary for the growth of certain bacteril viruses)liguofuliguofuliguofuliguofuFAre they enzymes? FFacilitated search (e.g., avoiding aggregates)? How? FCertainly, they do not specify the final
81、structure? FBut do they specify the path? FMay specify certain properties of the paths and/or intermediates? FMay provide an appropriate environment for folding? Many questions are yet to be answered. liguofuFive schemes of protein 3D structures1.The three-dimensional structure of a protein is deter
82、mined by its amino acid sequence.2.The function of protein depends on its structure.3.An isolated protein has a unique, or nearly unique, structure.4.The most important forces stabilizing the specific structure of a protein are non-covalent interactions.5.Amid the huge number of unique protein structures, we can recognize some common structural patterns to improve our understanding of protein architecture.SummaryliguofuSee you at “Protein Function”liguofu
