Two proteins with the same structure respond very differently to mutation: the role of plasticity in protein stability

As part of a systematic study of the folding of protein structural families we compare the effect of mutation in two closely related fibronectin type III (fnIII) domains, the tenth fnIII domain of human fibronectin (FNfn10) and the third fnIII domain of human tenascin (TNfn3). This comparison of the two related proteins allows us to distinguish any anomalous response to mutation. Although they have very similar structures, the effect of mutation is very different. TNfn3 behaves like a "typical" protein, with changes in free energy correlated to the number of contacts lost on mutation. The loss of free energy upon mutation is significantly lower for FNfn10, particularly mutations of residues in the A, B and G strands. Remarkably, some of the residues involved are completely buried and closely packed in the core. In FNfn10 the regions of the protein that can accommodate mutation have previously been shown to be mobile. We propose that there is a "plasticity" in the peripheral regions of FNfn10 that allows it to rearrange to minimise the effect of mutations. This study emphasises the difficulties that might arise when making generalisations from a single member of a protein family.     

Crosstalk between the protein surface and hydrophobic core in a core-swapped fibronectin type III domain

Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only ∼ 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the “outside” (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.     

The folding nucleus of a fibronectin type III domain is composed of core residues of the immunoglobulin-like fold

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Br?nsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is approximately 40 % of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different "layers" of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.     

Hydrophobic core fluidity of homologous protein domains: relation of side-chain dynamics to core composition and packing

The side-chain dynamics of methyl groups in two structurally related proteins from the fibronectin type III (fnIII) superfamily, the third fnIII domain from human tenascin (TNfn3) and the tenth fnIII domain from human fibronectin (FNfn10), have been studied by NMR spectroscopy. Side-chain order parameters reveal that the hydrophobic cores of the two proteins have substantially different mobilities. The core of TNfn3 is very dynamic, with exceptionally low order parameters for the most deeply buried residues, while that of FNfn10 is more like those of other proteins which have been studied with this technique, having a relatively rigid core with uniformly distributed dynamics. The unusually dynamic core of TNfn3 appears to be related to its amino acid composition, which makes it more fluid-like. A further explanation for the mobility of the TNfn3 core may be found in the negative correlation between the order parameter and excess packing volume, which shows that the core of TNfn3 is less densely packed and consequently has lower methyl order parameters for its buried residues. Rotameric transitions, presumably facilitated by the lower packing density, appear to make an important contribution to lowering the order parameters, and have been probed by measuring three-bond scalar couplings. Overall, although backbone dynamics is generally similar for proteins with the same topology on a fast time scale (picoseconds to nanoseconds), this study shows that a single fold can accommodate a wide variation in the dynamic properties of its core.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.     

Sequence conservation in Ig-like domains: the role of highly conserved proline residues in the fibronectin type III superfamily

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain-domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.     

Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.     

Refolding kinetics of cytochrome c(551) reveals a mechanistic difference between urea and guanidine

The energetic parameters for the folding of small globular proteins can be very different if derived from guanidine hydrochloride (GdnHCl) or urea denaturation experiments. A study of the equilibrium and kinetics of the refolding of wild-type (wt) cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa and of two site-directed mutants (E70Q and E70V) shows that the nonionic nature of urea reveals the role of a salt bridge between residues E70 and K10 on the transition state, which is otherwise completely masked in GdnHCl experiments. Mixed denaturant refolding experiments allow us to conclude that the masking effect of GdnHCl is complete at fairly low GdnHCl concentrations ( congruent with 0.1 M). The fact that potassium chloride is unable to reproduce this quenching effect, together with the results obtained on the mutants, suggests a specific binding of the Gdn(+) cation, which involves the E70-K10 ion pair in wt cyt c(551).We propose, therefore, a simple kinetic test to obtain a mechanistic interpretation of nonlinear dependences of DeltaG(w) on GdnHCl concentration on the basis of kinetic refolding experiments in the presence of both denaturants.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

Apparent Debye-Huckel electrostatic effects in the folding of a simple, single domain protein

We have monitored the effects of salts and denaturants on the folding of the simple, two-state protein FynSH3. As predicted by Debye-Huckel limiting law, both the stability and (log) folding rate of FynSH3 increase nearly perfectly linearly (r(2)> 0.99) with the square root of ionic strength upon increasing concentrations of the relatively nonchaotropic salt sodium chloride. The stability of FynSH3 is also linear in square root ionic strength when the relatively nonchaotropic salts sodium bromide, potassium bromide, and potassium chloride are employed. Comparison of the kinetic and equilibrium effects of sodium chloride suggests that the electrostatic interactions formed in the folding transition state are approximately 50% as destabilizing as those formed in the native state, presumably reflecting the more compact nature of the latter. In contrast, the relationship between concentration and folding kinetics is more complex when the highly chaotropic salt guanidine hydrochloride (GuHCl) is employed. At moderate to high GuHCl concentrations the net effect of the linear, presumably chaotrope-induced deceleration and the presumed, square root-dependent ionic strength-induced acceleration is well approximated as linear, thus accounting for the observation of "chevron behavior" (log folding rate linear in denaturant concentration) typically reported for the folding of single domain proteins. At very low GuHCl concentrations, however, significant kinetic rollover is observed. This rollover is reasonably well fitted as a sum of a linear, presumably chaotropic effect and a square root-dependent, presumably electrostatic effect. These results thus not only provide insight into the nature of the folding transition state but also suggest that caution is in order when extrapolating GuHCl-based chevrons to estimate folding rates in the absence of denaturant and in interpreting deviations from chevron linearity as evidence for non-two-state kinetics.     

Experiments suggest that simulations may overestimate electrostatic contributions to the mechanical stability of a fibronectin type III domain

Steered molecular dynamics simulations have previously been used to investigate the mechanical properties of the extracellular matrix protein fibronectin. The simulations suggest that the mechanical stability of the tenth type III domain from fibronectin (FNfn10) is largely determined by a number of critical hydrogen bonds in the peripheral strands. Interestingly, the simulations predict that lowering the pH from 7 to approximately 4.7 will increase the mechanical stability of FNfn10 significantly (by approximately 33 %) due to the protonation of a few key acidic residues in the A and B strands. To test this simulation prediction, we used single-molecule atomic force microscopy (AFM) to investigate the mechanical stability of FNfn10 at neutral pH and at lower pH where these key residues have been shown to be protonated. Our AFM experimental results show no difference in the mechanical stability of FNfn10 at these different pH values. These results suggest that some simulations may overestimate the role played by electrostatic interactions in determining the mechanical stability of proteins.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Revisiting the folding kinetics of bacteriorhodopsin

The elucidation of the physical principles that govern the folding and stability of membrane proteins is one of the greatest challenges in protein science. Several insights into the folding of α-helical membrane proteins have come from the investigation of the conformational equilibrium of H. halobium bacteriorhodopsin (bR) in mixed micelles using SDS as a denaturant. In an effort to confirm that folded bR and SDS-denatured bR reach the same conformational equilibrium, we found that bR folding is significantly slower than has been previously known. Interrogation of the effect of the experimental variables on folding kinetics reveals that the rate of folding is dependent not only on the mole fraction of SDS but also on the molar concentrations of mixed micelle components, a variable that was not controlled in the previous study of bR folding kinetics. Moreover, when the molar concentrations of mixed micelle components are fixed at the concentrations commonly employed for bR equilibrium studies, conformational relaxation in the transition zone is slower than hydrolysis of the retinal Schiff base. As a result, the conformational equilibrium between folded bR and SDS-denatured bR cannot be achieved under the conventional condition. Our finding suggests that the molar concentrations of mixed micelle components are important experimental variables in the investigation of the kinetics and thermodynamics of bR folding and should be accounted for to ensure the accurate assessment of the conformational equilibrium of bR without the interference of retinal hydrolysis.     

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain

The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.     

The kinetic pathway of folding of barnase

To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.