Presentation on theme: "Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure"— Presentation transcript:
1 Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure
2 Essential QuestionHow do the forces of chemical bonding determine the formation, stability, and myriad functions of proteins?
3 Outline What noncovalent interactions stabilize protein structure? What role does the amino acid sequence play in protein structure?What are the elements of secondary structure in proteins, and how are they formed?How do polypeptides fold into three-dimensional protein structures?How do protein subunits interact at the quaternary level of protein structure?
4 Protein Structure and Function Are Tightly Linked The three-dimensional structures of proteins and their biological functions are linked by several overarching principles:Function depends on structureStructure depends on sequence and on weak, noncovalent forcesThe number of protein folding patterns is large but finiteStructures of globular proteins are marginally stableMarginal stability facilitates motionMotion enables function
5 6.1 What Noncovalent Interactions Stabilize the Higher Levels of Protein Structure? Secondary, tertiary, and quaternary structure of proteins is formed and stabilized by weak forcesHydrogen bonds are formed wherever possibleHydrophobic interactions drive protein foldingIonic interactions usually occur on the protein surfacevan der Waals interactions are ubiquitous
6 Electrostatic Interactions in Proteins An electrostatic interaction between a positively charged lysine amino group and a negatively charged glutamate carboxyl group.
7 The atoms of the peptide bond lie in a plane 6.3 What Are the Elements of Secondary Structure in Proteins, and How Are They Formed?The atoms of the peptide bond lie in a planeAll protein structure is based on the amide planeThe resonance stabilization energy of the planar structure is 88 kJ/molA twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle.Rotation can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backbone
8 6.3 What Are the Elements of Secondary Structure in Proteins, and How Are They Formed? The amide or peptide bond planes are joined by the tetrahedral bonds of the α-carbon.The rotation parameters are φ and ψ. The conformations shown corresponds to φ= 180° and ψ= 180°.
9 Consequences of the Amide Plane Two degrees of freedom per residue for the peptide chainAngle about the Cα-N bond is denoted φ (phi)Angle about the Cα-C bond is denoted ψ (psi)The entire path of the peptide backbone is known if all φ and ψ angles are specifiedSome values of φ and ψ are more likely than others.
10 Some Values of φ and ψ Are Not Allowed Many of the possible conformations about an α-carbon between two peptide planes are forbidden because of steric crowding.
11 Steric Constraints on φ & ψ Unfavorable orbital overlap/steric crowding precludes some combinations of φ and ψφ = 0°, ψ = 180° is unfavorableφ = 180°, ψ = 0° is unfavorableφ = 0°, ψ = 0° is unfavorable
13 Classes of Secondary Structure Secondary structures are local structures that are stabilized by hydrogen bondsAlpha helicesOther helicesBeta sheet (composed of "beta strands")Tight turns (aka beta turns or beta bends)Beta bulge
14 The α-HelixFour different representations of the α-helix.
15 The α-Helix Numbers to Know Residues per turn: 3.6 Rise per residue: 1.5 Angstroms (0.15 nm)Rise per turn (pitch): 3.6 1.5Å = 5.4 AngstromsThe backbone loop that is closed by any H-bond in an alpha helix contains 13 atomsφ = −60 degrees, ψ = −45 degreesThe non-integral number of residues per turn was a surprise to crystallographers
16 The α-Helix in Proteins Two proteins that contain substantial amounts of α-helix.
17 Amino acids can be classified as helix-formers or helix breakers
18 The β-Pleated Sheet The β-pleated sheet is composed of β-strands Also first postulated by Pauling and Corey, 1951Strands in a β-sheet may be parallel or antiparallelRise per residue:3.47 Angstroms for antiparallel strands3.25 Angstroms for parallel strandsEach strand of a β-sheet may be pictured as a helix with two residues per turn
19 The β-Pleated SheetA “pleated sheet” of paper with an antiparallel β-sheet drawn on it.
20 The β-Pleated SheetH bonds in parallel and antiparallel β-sheets
21 Helix-Sheet Composites in Spider Silk Spider web silks are composites of α-helices and β-sheets. The radial strands of webs must be strong and rigid and have a higher percentage of β-sheets. The circumferential strands (termed capture silk) must be flexible and contain a higher percentage of α-helices.
22 (aka β-bend, or tight turn) The β-Turn(aka β-bend, or tight turn)Allows the peptide chain to reverse directionCarbonyl C of one residue is H-bonded to the amide proton of a residue three residues awayProline and glycine are prevalent in β-turnsThere are two principal forms of β-turns
23 The β-TurnThe structures of two kinds of β-turns (also called tight turns or β-bends). Four residues are required to form a β-turn. Left: Type I; right: Type II.
24 6.4 How Do Polypeptides Fold into Three-Dimensional Protein Structures? Several important principles:Secondary structures form wherever possible (due to formation of large numbers of H bonds)Helices and sheets often pack close togetherPeptide segments between secondary structures tend to be short and directProteins fold so as to form the most stable structures. Stability arises from:Formation of large numbers of intramolecular hydrogen bondsReduction in the surface area accessible to solvent that occurs upon folding
25 Two factors lie at the heart of these principles: 6.4 How Do Polypeptides Fold into Three-Dimensional Protein Structures?Two factors lie at the heart of these principles:Proteins are typically a mixture of hydrophilic and hydrophobic amino acidsThe hydrophobic groups tend to cluster together in the folded interior of the protein
26 Fibrous ProteinsMuch or most of the polypeptide chain is organized approximately parallel to a single axisFibrous proteins are often mechanically strongFibrous proteins are usually insolubleUsually play a structural role in natureThree types of fibrous protein are discussed here:α-Keratinβ-KeratinCollagen
27 α-KeratinA fibrous protein found in hair, fingernails, claws, horns and beaksSequence consists of residue alpha helical rod segments capped with non-helical N- and C-terminiPrimary structure of helical rods consists of 7-residue repeats: (a-b-c-d-e-f-g)n, where a and d are nonpolar.This structure promotes association of helices to form coiled coils
28 Collagen – A Triple Helix Principal component of connective tissue (tendons, cartilage, bones, teeth)Basic unit is tropocollagen:Three intertwined polypeptide chains (1000 residues each)MW = 285,000300 nm long, 1.4 nm diameterUnique amino acid composition, including hydroxylysine and hydroxyprolineHydroxyproline is formed by the vitamin C-dependent prolyl hydroxylase reaction.
29 Collagen – A Triple Helix The secrets of its a.a. composition...Nearly one residue out of three is GlyProline content is unusually highUnusual amino acids found:4-hydroxyproline3-hydroxyproline5-hydroxylysinePro and HyPro together make 30% of residues
30 Globular Proteins Mediate Cellular Function Globular proteins are more numerous than fibrous proteinsThe diversity of protein structures in nature reflects the remarkable variety of functions they performFunctional diversity derives in turn from:The large number of folded structures that polypeptides can adoptThe varied chemistry of the side chains of the 20 common amino acids
31 Some design principles Globular ProteinsSome design principlesHelices and sheets make up the core of most globular proteinsMost polar residues face the outside of the protein and interact with solventMost hydrophobic residues face the interior of the protein and interact with each otherPacking of residues is closeHowever, ratio of van der Waals volume to total volume is only 0.72 to 0.77, so empty space existsThe empty space is in the form of small cavities
32 “Random coils” are not random The segments of a protein that are not helices or sheets are traditionally referred to as “random coil”, although this term is misleading:Most of these segments are neither coiled or randomThey are usually organized and stable, but don’t conform to any frequently recurring patternRandom coil segments are strongly influenced by side-chain interactions with the rest of the protein
33 Globular ProteinsThe structure of ribonuclease, showing elements of helix, sheet and random coil.
34 Protein surfaces are complex The surfaces of proteins are complementary to the molecules they bind.
35 Waters on the Protein Surface Stabilize the Structure The surfaces of proteins are ideally suited to form multiple H bonds with water molecules.
36 α-Helices May be Polar, Nonpolar or Amphiphilic The so-called helical wheel presentation can reveal the polar or nonpolar character of α-helices.
37 Protein domains are nature’s modular strategy for protein design Proteins composed of about 250 amino acids or less often have a simple, compact globular shapeLarger globular proteins are typically made up of two or more recognizable and distinct structures, termed domains or modules – compact, folded protein structures that are usually stable by themselves in aqueous solutionDomains may consist of a single continuous portion of the protein sequence (see Figure 6.23)In some proteins, the domain sequence is interrupted by a sequence belonging to another part of the protein (Figure 6.24)
38 Many proteins are composed of several distinct domains Several protein modules used in the construction of complex multimodule proteins.
39 Classification Schemes for the Protein Universe Are Based on Domains Common features of SCOP and CATH:Class is determined from overall composition of secondary structure elements in a domainFold describes the number, arrangement, and connections of these secondary structure elementsSuperfamily includes domains of similar folds and usually similar functionsFamily usually includes domains with closely related amino acid sequences
40 Structure and Function are Not Always Linked Because structure depends on sequence, and because function depends on structure, it is tempting to imagine that all proteins of similar structure should share a common function, but this is not always trueSome proteins of similar domain structure have different functionsSome proteins of similar function possess very different structures
42 Denaturation Leads to Loss of Protein Structure and Function The cellular environment is suited to maintaining the weak forces that preserve protein structure and functionExternal stresses – heat, chemical treatment, etc. – can disrupt these forces in a process termed denaturation – the loss of structure and functionThe cooking of an egg is an everyday exampleOvalbumin, the principal protein in egg white, remains in its native structure up to a characteristic melting temperature, TmAbove this temperature, the structure unfolds and function is lost
43 Denaturation Leads to Loss of Protein Structure and Function The proteins of egg white are denatured during cooking. More than half of the protein in egg white is ovalbumin.
44 Denaturation Leads to Loss of Protein Structure and Function Proteins can be denatured by heat, with commensurate loss of function.
45 Denaturation Leads to Loss of Protein Structure and Function Proteins can be denatured (unfolded) by high concentrations of guanidine-HCl or urea. The denaturation of chymotrypsin is plotted here.
46 Anfinsen’s Classic Experiment Proved that Sequence Determines Structure Ribonuclease can be unfolded by treatment with urea β-Mercaptoethanol (MCE) cleaves disulfide bonds. Anfinsen showed that ribonuclease structure (and function) could be restored under appropriate conditions.
47 Is There a Single Mechanism for Protein Folding? How a protein achieves its stable, folded state is a complex questionLevinthal’s paradox demonstrates that proteins cannot fold by sampling all possible conformationsThis implies that proteins actually fold via specific “folding pathways”What factors play a role in protein folding processes?
48 Postulated Themes of Protein Folding Secondary structures – helices, sheets, and turns – probably form firstNonpolar residues may aggregate or coalesce in a process termed a hydrophobic collapseSubsequent steps probably involve formation of long-range interactions between secondary structures or involving other hydrophobic interactionsThe folding process may involve one or more intermediate states, including transition states and what have become known as molten globules
49 The Protein Folding Energy Landscape Ken Dill has suggested that the folding process can be pictured as a funnel of free energies. The rim at the top represents the many unfolded states. Polypeptides ‘fall down the wall of the funnel’ to ever fewer possibilities and lower energies as they fold.
50 Motion is Important for Globular Proteins Protein are dynamic structures – they oscillate and fluctuate continuously about their average or equilibrium structuresThis flexibility is essential for protein functions, including:Ligand bindingEnzyme catalysisEnzyme regulation
51 Motion is Important for Globular Proteins Proteins are dynamic structures. The marginal stability of a tertiary structure leads to flexibility and motion in the protein.
52 Most Globular Proteins Belong to One of Four Structural Classes Proteins can be classified according to the type and arrangement of secondary structureThere are four classes:All α proteins, in which α helices predominateAll β proteins, in which β sheets predominateα/β proteins, in which helices and sheets are intermingledα+β proteins, which contain separate α-helical and β-sheet domains
53 Most Globular Proteins Belong to One of Four Structural Classes Four major classes of protein structure (as defined in the SCOP database).
54 Molecular Chaperones Are Proteins That Help Other Proteins to Fold Why are chaperones needed if the information for folding is inherent in the sequence?to protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow stepsChaperone proteins were first identified as "heat-shock proteins" (Hsp60 and Hsp70)
55 Some Proteins Are Intrinsically Unstructured Many proteins exist and function normally in a partially unfolded stateThese intrinsically unstructured proteins (IUPs) do not possess uniform structural properties but are still essential for cellular functionThese proteins are characterized by a nearly complete lack of structure and high flexibilityIUPs adopt well-defined structures in complexes with their target proteinsIUPs are characterized by an abundance of polar residues and a lack of hydrophobic residues
56 Some Proteins Are Intrinsically Unstructured Intrinsically unstructured proteins (IUPs) contact their target proteins over a large surface area.
57 α1-Antitrypsin – A Tale of Molecular Mousetraps and a Folding Disease α1-Antitrypsin normally blocks elastase in the lungsIt functions as a molecular mousetrap, binding elastase, then dragging the bound elastase to the other side of the antitrypsinAt this new site, elastase is inactivated and degradedDefects in α1-antitrypsin can result in lung and liver damageGenetic variants are often inactiveIn smokers, oxidation of a crucial Met in the flexible loop also inactivates α1-antitrypsin, leading to emphysema
58 α1-Antitrypsin – A Tale of Molecular Mousetraps and a Folding Disease Elastase is inactivated by binding to α1-antitrypsin
59 Diseases of Protein Folding A number of human diseases are linked to abnormalities of protein foldingProtein misfolding may cause disease by a variety of mechanismsMisfolding may result is loss of function and the onset of diseaseThe table on the next slide summarizes some known protein folding disease
61 What are the forces driving quaternary association? 6.5 How Do Protein Subunits Interact at the Quaternary Level of Structure?What are the forces driving quaternary association?Typical Kd for two subunits: 10−8 to 10−16M!These values correspond to energies of kJ/mol at 37° CEntropy loss due to association - unfavorableEntropy gain due to burying of hydrophobic groups - very favorable!
62 6.5 How Do Protein Subunits Interact at the Quaternary Level of Structure? The quaternary structure of liver alcohol dehydrogenase.
63 6.5 How Do Protein Subunits Interact at the Quaternary Level of Structure? The subunit compositions of several proteins. Proteins with two or four subunits predominate in nature, and many cases of higher numbers exist.
64 6.5 How Do Protein Subunits Interact at the Quaternary Level of Structure? Figure Multimeric proteins are symmetric arrangements of asymmetric objects. A variety of symmetries is displayed in these multimeric structures.
65 QuestionsYou should be able to complete questions 1-4, 6-13 at the end of the chapter.