Structural comparison of the two alternative transition states for folding of TI I27

TI I27, a beta-sandwich domain from the human muscle protein titin, has been shown to fold via two alternative pathways, which correspond to a change in the folding mechanism. Under physiological conditions, TI I27 folds by a classical nucleation-condensation mechanism (diffuse transition state), whereas at extreme conditions of temperature and denaturant it switches to having a polarized transition state. We have used experimental Phi-values as restraints in ensemble-averaged molecular dynamics simulations to determine the ensembles of structures representing the two transition states. The comparison of these ensembles indicates that when native interactions are substantially weakened, a protein may still be able to fold if it can access an alternative transition state characterized by a much larger entropic contribution. Analysis of the probability distribution of Phi-values derived from ensemble averaged simulations, enables us to identify residues that form contacts in some members of the ensemble but not in others illustrating that many interactions present in transition states are not strictly required for the successful completion of the folding process.     

Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump

Small proteins often fold in an apparent two-state manner with the absence of detectable early-folding intermediates. Recently, using native-state hydrogen exchange, intermediates that exist after the rate-limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post-transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser-induced temperature-jump (T-jump) technique to investigate this issue and have applied it to Rd-apocyt b(562) , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b(562) folds via an on-pathway hidden intermediate, which has only the N-terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N-terminal helix of Rd-apocyt b(562) was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two-state folding behavior. However, in the T-jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd-apocyt b(562) . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 microsec)(-1) at 65 degrees C.     

Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (C(L)), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric C(L) domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr(141)-Pro(142) bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.     

Phi-analysis of the folding of the B domain of protein A using multiple optical probes

We examined the co-operativity of ultra-fast folding of a protein and whether the Phi-value analysis of its transition state depended on the location of the optical probe. We incorporated in turn a tryptophan residue into each of the three helices of the B domain of Protein A. Each Trp mutant of the three-helix bundle protein was used as a pseudo-wild-type parent for Phi-analysis in which the intrinsic Trp fluorescence probed the formation of each helix during the transition state. Apart from local effects in the immediate vicinity of the probe, the three separate sets of Phi-values were in excellent agreement, demonstrating the overall co-operativity of folding and the robustness of the Phi-analysis. The transition state of folding of Protein A contains the second helix being well formed with many stabilizing tertiary hydrophobic interactions. In contrast, the first and the third helices are more poorly structured in the transition state. The mechanism of folding thus involves the concurrent formation of secondary and tertiary interactions, and is towards the nucleation-condensation extreme in the nucleation-condensation-framework continuum of mechanism, with helix 2 being the nucleus. We provide an error analysis of Phi-values derived purely from the kinetics of two-state chevron plots.     

Fast and faster: a designed variant of the B-domain of protein A folds in 3 microsec

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 microsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.     

Key role of coulombic interactions for the folding transition state of the cold shock protein

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Population of on-pathway intermediates in the folding of ubiquitin

The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

A framework for describing topological frustration in models of protein folding

In a natively folded protein of moderate or larger size, the protein backbone may weave through itself in complex ways, raising questions about what sequence of events might have to occur in order for the protein to reach its native configuration from the unfolded state. A mathematical framework is presented here for describing the notion of a topological folding barrier, which occurs when a protein chain must pass through a hole or opening, formed by other regions of the protein structure. Different folding pathways encounter different numbers of such barriers and therefore different degrees of frustration. A dynamic programming algorithm finds the optimal theoretical folding path and minimal degree of frustration for a protein based on its natively folded configuration. Calculations over a database of protein structures provide insights into questions such as whether the path of minimal frustration might tend to favor folding from one or from many sites of folding nucleation, or whether proteins favor folding around the N terminus, thereby providing support for the hypothesis that proteins fold co-translationally. The computational methods are applied to a multi-disulfide bonded protein, with computational findings that are consistent with the experimentally observed folding pathway. Attention is drawn to certain complex protein folds for which the computational method suggests there may be a preferred site of nucleation or where folding is likely to proceed through a relatively well-defined pathway or intermediate. The computational analyses lead to testable models for protein folding.     

The Folding Pathway of the Antibody VL Domain

Antibodies are modular proteins consisting of domains that exhibit a β-sandwich structure, the so-called immunoglobulin fold. Despite structural similarity, differences in folding and stability exist between different domains. In particular, the variable domain of the light chain V_L is unusual as it is associated with misfolding diseases, including the pathologic assembly of the protein into fibrillar structures. Here, we have analysed the folding pathway of a V_L domain with a view to determine features that may influence the relationship between productive folding and fibril formation. The V_L domain from MAK33 (murine monoclonal antibody of the subtype κ/IgG1) has not previously been associated with fibrillisation but is shown here to be capable of forming fibrils. The folding pathway of this V_L domain is complex, involving two intermediates in different pathways. An obligatory early molten globule-like intermediate with secondary structure but only loose tertiary interactions is inferred. The native state can then be formed directly from this intermediate in a phase that can be accelerated by the addition of prolyl isomerases. However, an alternative pathway involving a second, more native-like intermediate is also significantly populated. Thus, the protein can reach the native state via two distinct folding pathways. Comparisons to the folding pathways of other antibody domains reveal similarities in the folding pathways; however, in detail, the folding of the V_L domain is striking, with two intermediates populated on different branches of the folding pathway, one of which could provide an entry point for molecules diverted into the amyloid pathway.     

The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family

Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Φ-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Φ-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Φ values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Φ values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains.     

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.     

Downhill versus Barrier-Limited Folding of BBL: 3. Heterogeneity of the Native State of the BBL Peripheral Subunit Binding Domain and Its Implications for Folding Mechanisms

Protein folding studies are generally predicated on Anfinsen's dogma that there is a unique native state of a protein. However, this is not always the case. NMR measurements of BBL, for example, find a decrease in helicity of helix 2 surrounding His166 on its protonation, which, with other experimental data, suggests that the native state can occupy two or more conformations. Here, we analysed the native structure of BBL as a function of pH, temperature and ionic strength, along with a truncated BBL construct, by extensive all-atom molecular dynamics simulations in explicit solvent, corresponding to at least 400 ns of trajectories collected for each set of conditions. The native state was heterogeneous under a variety of conditions, consisting of two predominant conformations. This equilibrium changed with conditions: protonation of His166 at low pH shifted the equilibrium in favour of a less ordered conformer, while high ionic strength at neutral pH shifted the equilibrium to a more ordered conformer. Furthermore, high temperature and truncation of the sequence also shifted the equilibrium toward the less ordered conformer. Importantly, conformational heterogeneity in a native structure that changes with conditions will lead to deviations from the classic two-state behaviour during the barrier-limited unfolding of a protein. In particular, some regions of the protein will appear to unfold asynchronously and some residues will have anomalous thermal titration curves and unusual baseline behaviour monitored microscopically by NMR spectroscopy and macroscopically by calorimetry and other techniques. Such data could otherwise be interpreted as evidence for barrier-free downhill folding. Any biological significance of downhill folding of BBL appears to be ruled out by recent crystallographic studies on the reaction cycle of the BBL-equivalent domain in a pyruvate dehydrogenase multienzyme complex in which the domain remains of constant structure.     

Thermostability of temperature-sensitive folding mutants of the P22 tailspike protein

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37-42 degrees C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 degrees C, the melting temperatures of the mutant proteins were all above 82 degrees C, more than 40 degrees C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo, the thermostability of the native protein was also examined with infected cells. Once matured at 28 degrees C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 degrees C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.     

The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.     

Rapid cooperative two-state folding of a miniature alpha-beta protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.     

Folding of the striated muscle myosin motor domain

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.     

Unfolding of the cold shock protein studied with biased molecular dynamics

The cold shock protein from Bacillus caldolyticus is a small beta-barrel protein that folds in a two-state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native-like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three-stranded beta-sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two-stranded beta-strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them.