Protein Stability and Folding - Lecture Slides | CHEM 660, Study notes of Chemistry

Download Protein Stability and Folding - Lecture Slides | CHEM 660 and more Study notes Chemistry in PDF only on Docsity! Protein Stability and Folding Stability of the Folded Conformation • Folded conformation is only marginally stable and can be disrupted by changes in environment (heat, pH, increased pressure or addition of denaturants). • Protein denaturation need not involve changes in covalent structure and is usually reversible. • As the environment changes towards denaturing conditions, initially the structure of small single-domain proteins changes very little. • May be increases in flexibility, but overall structure is unchanged. • The protein then denatures entirely over a very narrow range. • This abrupt unfolding is indicative of a very cooperative transition. • The unfolding of most small single-domain proteins is reversible and equilibrium can be attained. • Many methods available for visualizing/monitoring unfolding. Structure 1320 Figure 1. Design of Ala2Ile2-6 (a) An !-helical wheel diagram (looking down the long axis) of the heptad repeat of Rop. The “a” (yellow) and “d” (red) residues form the hydrophobic core and are the residues mutated in the repacked protein Ala2Ile2-6. Two layers of the core are shown. Helices from protomer A are designated 1 and 2, and the protomer B helices are labeled 1" and 2". Arrows indicate the direction of the polypeptide chain from the N terminus to the C terminus. (b) Sequence alignment of Rop and Ala2Ile2-6 with the residue number placed above every tenth residue. The “a” and “d” residues are colored to match the diagram. To create Ala2Ile2-6, residues in the “a” and “d” positions of Rop were changed to alanine and isoleucine, respectively. The outermost layer at each end of the four-helix bundle, consisting of residues 5, 29, 31, and 56, were not changed. Residue 56, in the “e” position of the heptad repeat, acts as a “d” residue by packing its side chain into the appropriate core position. Figure 2. Thermodynamic Comparison of Rop and Ala2Ile2-6 (a) Thermal stability profile (#G versus T) and representative thermal denatur- ations (inset) of Rop (solid circles) and Ala2Ile2-6 (open circles). (b) Calculated values of #H (squares) and -T#S (circles) as a function oflost RNA binding activity, but which has enhanced, nativelike temperature for Rop (solid) and Ala2Ile2-6 (open).thermal stability. Results and Discussion chain volume and hydrophobicity, any structural and thermo- dynamic differences between Ala2Leu2-6 and Ala2Ile2-6 are,Design and Initial Characterization Rop variants were generated by replacing the core residues therefore, primarily a consequence of the different side chain stereochemistries of leucine and isoleucine.(Figure 1a) of the heptad repeat with a regular pattern of hy- drophobic amino acids. One of the first and most conservative The initial characterization of Ala2Ile2-6 demonstrated that it is highly helical with a circular dichroism (CD) spectrum similarmutants created was Ala2Leu2-6 [9], in which the middle six layers of the hydrophobic core incorporate alanines in the “a” to that of wild-type Rop. Furthermore, Ala2Ile2-6 maintained nativelike thermodynamic properties illustrated, for example,positions and leucines in the “d” positions. Ala2Leu2-6 has nativelike structural and thermodynamic properties, binds RNA by a cooperative and reversible thermal unfolding transition (Figure 2a, inset) accompanied by a large change in the heatwith wild-type affinity, and has a significantly higher melting temperature than that of wild-type Rop. To investigate the capacity (Table 1). Ala2Ile2-6 was shown to be a dimer by both sedimentation equilibrium centrifugation and multiangle laserimportance of side chain geometry in packing the core of Rop, we created Ala2Ile2-6 with alanine in the “a” positions and iso- light scattering measurements (data not shown). However, the electromobility shift assay for protein-RNA interaction [25]leucine in the “d” positions of the middle six layers of the core (Figure 1b). Because isoleucine and leucine share similar side demonstrated that Ala2Ile2-6 had completely lost the ability to Protein Folding: a Two-state Phenomenon • Protein folding/unfolding is a two-state phenomenon with only fully folded (N) and fully unfolded (U) protein states being present. Partially folded states are very unstable relative to N and U states. • For a two-state transition, the equilibrium constant between N and U can be measured directly from the average fraction of unfolding (!) in the transition region. • The value of Keq can be determined when ! is significantly different from 1 or 0 (in the transition region). • Allows calculation of "G under the set conditions (difference in free energy between U and N states). • When Keq = 1, "G = 0, provides a reference for calculating "G at other temperatures. • van’t Hoff analysis: uses temperature dependence of Keq to estimate "H and "S. Tm Keq = [U] [N] = 1 - ! ! "G = GN - GU = -RTlnKeq N U k1 k2 fraction of unfolded protein "G = "H - T"S "G(Tm)= 0 = "Hm - Tm"Sm ! = yU - yN y - yN • The heat capacity (Cp) is defined as the change in enthalpy (H) with temperature T. • Cp of the unfolded protein, Cp(U), is greater than that of the folded protein Cp(N). (difference between the two is the "Cp). • The Cp of proteins are dominated by the nonpolar surface area exposed to water. • It is possible to calculate "G at any temperature if one knows "Cp and the "H for a protein. • "H is most easily estimated at the Tm. • At high and low temperatures "G is dominated by the entropic component. !H / !T = Cp(U) - Cp(N) = "Cp Cp = !H / !T = T!S / !T "H(T2) = "H(T1) + "Cp(T2-T1) "G(Tm) = 0 = "Hm(Tm) - Tm "Sm "G(T) = "Hm(1-T/Tm) - "Cp[(Tm-T)+ T ln(T/Tm )] Structure 1320 Figure 1. Design of Ala2Ile2-6 (a) An !-helical wheel diagram (looking down the long axis) of the heptad repeat of Rop. The “a” (yellow) and “d” (red) residues form the hydrophobic core and are the residues mutated in the repacked protein Ala2Ile2-6. Two layers of the core are shown. Helices from protomer A are designated 1 and 2, and the protomer B helices are labeled 1" and 2". Arrows indicate the direction of the polypeptide chain from the N terminus to the C terminus. (b) Sequence alignment of Rop and Ala2Ile2-6 with the residue number placed above every tenth residue. The “a” and “d” residues are colored to match the diagram. To create Ala2Ile2-6, residues in the “a” and “d” positions of Rop were changed to alanine and isoleucine, respectively. The outermost layer at each end of the four-helix bundle, consisting of residues 5, 29, 31, and 56, were not changed. Residue 56, in the “e” position of the heptad repeat, acts as a “d” residue by packing its side chain into the appropriate core position. Figure 2. Thermodynamic Comparison of Rop and Ala2Ile2-6 (a) Thermal stability profile (#G versus T) and representative thermal denatur- ations (inset) of Rop (solid circles) and Ala2Ile2-6 (open circles). (b) Calculated values of #H (squares) and -T#S (circles) as a function oflost RNA binding activity, but which has enhanced, nativelike temperature for Rop (solid) and Ala2Ile2-6 (open).thermal stability. Results and Discussion chain volume and hydrophobicity, any structur l and thermo- dynamic differences between Ala2Leu2-6 and Ala2Ile2-6 are,Design and Initial Characterization Rop variants were generated by replacing the core residues therefore, primarily a consequence of the different side chain stereochemistries of leucine and isoleucine.(Figure 1a) of the heptad repeat with a regular pattern of hy- drophobic amino acids. One of the first and most conservative The initial characterization of Ala2Ile2-6 demonstrated that it is highly helical with a circular dichroism (CD) spectrum similarmutants created was Ala2Leu2-6 [9], in which the middle six layers of the hydrophobic core incorporate alanines in the “a” to that of wild-type Rop. Furthermore, Ala2Ile2-6 maintained nativelike thermodynamic properties illustrated, for example,positions and leucines in the “d” positions. Ala2Leu2-6 has nativelike structural and thermodynamic properties, binds RNA by a cooperative and reversible thermal unfolding transition (Figure 2a, inset) accompanied by a large change in the heatwith wild-type affinity, and has a significantly higher melting temperature than that of wild-type Rop. To investigate the capacity (Table 1). Ala2Ile2-6 was shown to be a dimer by both sedimentation equilibrium centrifugation and multiangle laserimportance of side chain geometry in packing the core of Rop, we created Ala2Ile2-6 with alanine in the “a” positions and iso- light scattering measurements (data not shown). However, the electromobility shift assay for protein-RNA interaction [25]leucine in the “d” positions of the middle six layers of the core (Figure 1b). Because isoleucine and leucine share similar side demonstrated that Ala2Ile2-6 had completely lost the ability to Protein Folding: a Two-state Phenomenon Physical Interactions • The unique properties of proteins is inextricably linked to the complex three-dimensional folded conformations they assume. • The three-dimensional folded conformation is the result of many simultaneous noncovalent interactions between different parts of the protein and with the environment. • These interactions are the result of a limited set of fundamental noncovalent forces. • The complexity of water and an aqueous environment limits our understanding of proteins. Protein Stability • Native proteins are only marginally stable under physiological conditions. • The free energy of denaturation is only ~0.1 kcal/mol for each amino amino acid residue. The folded conformation of a 100-residue protein would be ~10Kcal/mol more stable than the unfolded state. (energy required to break a hydrogen bond is 2-10Kcal/mol) • The three-dimensional folded conformation arises through a delicate balance of stabilizing and destabilizing forces. • The observed stability of the folded protein is the result of a very small difference between very large but compensating factors (enthalpy and entropy: both of which are temperature dependent). • The enthalpic and entropic contributions vary similarly and compensate each other. This results in the free energy being relatively small difference between the two. Forces Stabilizing Macromolecular Structure • Noncovalent interactions are key biological forces: ! Electrostatic Forces: # Ionic interactions # Van der Waals # Hydrogen bonding ! Hydrophobic interactions • These forces are transient in nature. • Several factors influence the strength of these interactions. • Individually all are weak (C-C bond ~80 Kcal/mol), but they add up and collectively can be very strong. Short-Range Repulsions • Repulsion eventually occurs between two molecules or atoms as they approach each other. • Repulsion invariably arises as they molecules/ atoms become near enough for their respective electron orbitals begin to overlap. • Repulsion increases enormously because the electrons on the different molecules cannot occupy the same space at the same time (increasing exponentially with the inverse of distance). • Because repulsion rises so steeply, it is possible to consider molecules/atoms to have definite dimensions with defined volumes (van der Waals radius). • van der Waals radius is based on smallest distance that can exist between two nonbonded atoms in the crystalline state. • Accessible surface area provides a more practical concept of surface for proteins and other biomacromolecules (described by center of a water molecule with radius 1.4 Å in van der Waals contact with the molecule). Electrostatic Forces: Point Charges • All intermolecular forces are thought to be essentially electrostatic in origin. • The most fundamental noncovalent interaction would therefore be the interaction between electrostatic charges. • Coulomb’s law describes the interaction between two point charges in a vacuum. • It describes an interaction that is effective over relatively long distances. • For other environments (such as in solution) the electrostatic interaction is modulated by other interactions. • In homogenous environments, the electrostatic interaction is diminished by the dielectric constant of the medium (dielectric constant of water = ~80). • At short distances, molecules and atoms cannot be treated as point charges. • Electrostatic effects in proteins involve changes in their ionization tendencies, pKa values. • Interactions between very close, oppositely charged groups in proteins usually involve not only electrostatic interactions, but also some degree of hydrogen bonding !E = ZA ZB " 2 rAB !E = ZA ZB " 2 rABD ZA = number of charges on A " = the charge of an electron ZB = number of charges on B rAB= distance between A and B D = dielectric constant Coulomb's Law (vacuum) Adjusted for environment • A molecule does not need to have a net charge to participate in electrostatic interactions. • Electron densities can be localized if covalently linked atoms have different electronegativities. " Atoms with a greater electronegativity have a partial negative charge ($-). " Atoms with a lower electronegativity have a partial positive charge ($+). • The separation of charge in a molecule determines its dipole moment (%D), corresponding to the magnitude of the separated charge (Z) and the distance d by which it is separated. • The dipole moment has directionality as well as magnitude. • The peptide bond which has partial double bond character exemplifies this polarization. The oxygen has a partial negative charge and the -NH- group a partial positive. • Dipoles interact with point charges, other dipoles and more complex interactions. µD = Zd C N C! O C! H C N C! O- C! H+ C N C! O C! H "- "+ +0.20 +0.42 -0.42 -0.20 Electronegativities of common atoms in proteins: O = 3.45 N = 2.98 C = 2.55 S = 2.53 H = 2.13 Dipole Moment Electrostatic Forces: van der Waals Hydrophobic Interactions • The magnitude of the hydrophobic interaction is generally measured by the free energy of transfer ("Gtr) of a nonpolar molecule in the gas, liquid or solid state into water. (positive "Gtr value indicates that the nonpolar molecule prefers a nonaqueous environment) • Transferring a solute molecule into a liquid involves: • Creating a suitable cavity in the liquid. • Introducing the solute molecule into the cavity. • Rearranging the solute and liquid molecules to maximize favorable interactions between them. • The observed thermodynamics of this transition are the net effect of all these factors. Interpretation is not always straightforward. • At room temperature, the unfavorable transfer of a nonpolar molecule from a nonpolar liquid to water is primarily a result of the unfavorable change in entropy ("Htr & 0). • The unfavorable entropy change is thought to result from increased ordering of water molecules around the nonpolar group. Surrounding water molecules appear to be more tightly packed than those of normal bulk water. • Water molecules cannot form hydrogen bonds with the nonpolar group. Therefore, they are generally believed to satisfy their hydrogen bond potential by forming a hydrogen bonded “iceberg” network among themselves at the nonpolar surface. Hydrophobic Interactions and Cp • As the temperature is increased, the ordered water shell around the nonpolar species tends to “melt” and become more like bulk water. This “melting” process requires energy resulting in the observed large heat capacity (Cp). • A large Cp is characteristic of aqueous solutions of nonpolar molecules, and is generally proportional to the exposed nonpolar surface area of the solute. • The Cp defines the temperature dependence of both entropy and enthalpy. • The temperature dependence of the hydrophobic interaction can provide insights into its physical nature. • At T>TH –– the entropy of transfer to water decreases, but "Htr becomes unfavorable. • At TS the "Str becomes 0 (TS thought to be ~140°C). • Large changes with temperature of "Htr and "Str mostly compensate and the value of "Gtr changes much less than they do. • It is important to specify the measure of hydrophobicity being used ("Gtr or Ktr). • At the high temperature Ts the persistence of low solubility of nonpolar groups in water is observed to be due to much weaker enthalpic interactions between the nonpolar species and water than between the nonpolar groups and the nonpolar solvent and between the water molecules in bulk water. Hydrophobic Interactions: to summarize • Hydrophobic interactions do not result from repulsion between water molecules and nonpolar molecules and surfaces. • While favorable interactions do occur between water molecules and nonpolar molecules and surfaces, the magnitude of these interactions are less than the favorable van der Waals interactions in a nonpolar environment and the hydrogen bonding in liquid water. • Hydrophobic interaction results in nonpolar atoms, molecules and groups to interact with each other rather than with water. Intramolecular Interactions • For molecules to interact with each other they must lose entropy, which is energetically unfavorable. • The magnitude of the loss in entropy is dependent on the degrees of freedom that become fixed as a result of the interaction. • In the case of intramolecular interactions, the groups involved are incorporated within the same molecular scaffold, which automatically limits the number of degrees of freedom by fixing the relative distance and orientation of the groups involved. • Intramolecular and bimolecular interactions can be compared by means of the ratio of their equi l ibr ium constants ( the ef fective concentration). A + B A•B Kinter A•B Kintra A B Kintra Kinter = effective concentration of A–B Intramolecular Interactions • The maximum effective concentration of two groups in an aqueous solution was believed to be 55M, but much greater values for effective concentration are usually observed. • Covalently linking the interacting moieties through a bond network results in their concentration relative to each other to be much higher than would be possible were the two groups on separate molecules. • Interaction between the two groups results in the sacrifice of some fraction of the internal flexibility and conformational freedom of the molecule. • When there is no entropic difference between molecules with and without interaction between the groups, their effective concentration is at its maximum value. " Interactions requiring proximity and orientation have very high maximum effective concentrations. " Interactions that are more tolerant and allow greater degrees of freedom have lower maximum effective concentrations. A + B A•B Kinter A•B Kintra A B Kintra Kinter = effective concentration of A–B Cooperativity of Multiple Interactions • Multiple groups within a single molecule can behave differently from the same groups in solution. • The simultaneous presence of multiple interactions within a single molecule results in cooperativity between them. Collectively, these interactions can be much stronger than expected based on their individual strengths. • Cooperativity is critical for proteins. • Single interactions between groups within a polypeptide chain are not expected to be stable unless these groups lie in close proximity of each other within the covalent structure (resulting in a high effective concentration). • Due to to the size and conformational flexibility of the unfolded protein, groups attached to a moderate sized peptide have effective concentrations in the range of 10-2-10-5 M (depending on proximity). • Expected values for Kobs,u (observed equilibrium constant) for individual hydrogen bonds, salt bridges... etc range from 4x10-3 to 10-7. A B • A B KAB[A/B]u Unfolded Folded Kobs,U = KAB[A/B]u [A/B]u = effective concentraoin of A and B in unfolded peptide KAB = association constant for free A and B Kinetic Analysis of Complex Reactions Kinetics of Unfolding • Protein unfolding is almost always observed to be an all or none process. • No partially folded intermediates are observed. • Native protein represents a relatively conformationally homogeneous population, and unfolding generally proceeds with a single kinetic phase and a single rate constant. (no lag phase). • Rate of unfolding generally changes with unfolding conditions. Kinetics of Refolding • Kinetic complexity is a hallmark of protein folding. Starting with the conformational heterogeneity of the unfolded population. • Heterogeneity includes cis-trans isomerization of peptide bonds (slow process). • In the folded protein, a peptide bond is usually cis or trans depending on which is favored by the protein conformation. • In the unfolded polypeptide, constraints favoring cis vs trans are released and an equilibrium between the isomers is attained at each peptide bond. • Refolding in the absence of slow peptide bond isomerization. • In an unfolded population with the native cis-trans isomers, refolding generally occurs with a single rate constant in spite of the conformational heterogeneity of the unfolded state. Kinetic Analysis of Protein Folding • Experimental approaches to study protein folding/ unfolding kinetics involve introducing unfolded protein into conditions that promote folding (with rapid mixing). • CD or fluorescence make it possible to follow folding on time scales ranging from 1 ms to ~10 min. • Modern instruments use pressure driven syringes to force reactants into mixing cell displacing the resident solution, which initiates monitoring of folding (stopped flow method) with a dead time of ~1 ms. • Kinetic profiles usually follow single exponential decays, and fitting the data provides kobs and maximum amplitude (Ai). • For a two-state system kinetic analysis is relatively straight forward. • Plotting the lnkobs against [denat] yields linear relationships for kf and ku (chevron plot). In chevron plots Cm is defined as the point where kf and ku are equivalent. kf and ku in the absence of denaturant is are extrapolated by extending each limb of the chevron plot to 0 denaturant. Chevron Plot kobs = Aie-kt Keq = kof/kou lnkf = lnkof +mf[denat] lnku = lnkou +mu[denat] kobs = kf +ku kobs = kou e(mu[denat]) + kof e(mf[denat])) mf and mu are kinetic m values. S(t) =S0-Ai(1-e-kobst) k= Ae-"G‡/RT Arrhenius Equation Hierarchal Protein Folding • The folding process begins with the formation of marginally stable local order/structure. • These structure elements then interact (locally) to form intermediates of increasing complexity. • Process continues ultimately yielding the native protein. • Evidence supporting the premise of hierarchal protein folding: # Many peptide fragments excised from proteins will assume their native conformation. # Observed folding intermediates are consistent with a hierarchal folding process. # Helix boundaries are fixed by their primary sequence, not so much by 3-D interactions. # Secondary structure can be predicted with reasonable accuracy, even when long-range interactions are not accounted for or are suppressed. • Sequence information defining a specific fold is both distributed throughout the polypeptide chain and is highly overdetermined. Protein Folding Events • Initial folding events (burst phase) • [milliseconds] $ For many small-single domain proteins, much of the secondary structure is established. $ Much of the driving force attributed to hydrophobic collapse. (hydrophobic groups coalesce and expel water) $ Initial collapsed state is molten globular. $ Side chains are extensively disordered. • Intermediate folding events: • [~5-1000 milliseconds] $ Secondary structure stabilizes and native-like tertiary structure appears. $ Side chains are still mobile. • Final folding events: • [! several seconds] $ Protein achieves native structure. $ Complex motions allow the protein to attain relatively rigid packing and hydrogen bonding. $ Remaining interior water molecules are expelled from the core. Landscape Theory of Protein Folding • Current Thinking: Protein folding is envisioned to proceed on an energy surface/landscape. • The landscape represents the conformational energy states available to a polypeptide. • Po l y p e p t i d e s f o l d v i a a s e r i e s o f conformational adjustments that reduce their free energy and entropy until the native folded state is achieved. • There is no single pathway or closely related set of pathways that a polypeptide must follow in achieving its native conformation. • Suggests that landscape maw include local energy minima and maxima (therefore many possible transient folding intermediates may exist) In cr ea si ng E ne rg y Decreasing Conformational Freedom Folding of Multidomain and Multimeric Proteins • Large proteins may be composed of multiple domains or polypeptide chains. • Independent domains unfold and refold like single-domain proteins, which can lead to complex unfolding curves for proteins. (in such cases, domains may unfold under different conditions) • Can also be varying degrees of interaction between the domain. Interactions between domains can effect folding. • Where the isolated domains are stable, folding of the intact multidomain protein appears to occur by initial folding of the domains, followed by association of the domains. • Domain association is often the slowest step in the folding process. (domains may not be folded entirely correctly or because small adjustments are required for interaction between the domains.) • When association is slow step, an intermediate can accumulate where domains are folded but impaired. May lead to intermolecular interactions and precipitation.