Major Transition State Research Articles

Simulations of the structural and dynamical properties of denatured proteins: the “molten coil” state of bovine pancreatic trypsin inhibitor

The dynamic nature of denatured, unfolded proteins makes it difficult to characterize their structures experimentally. To complement experiment and to obtain more detailed information about the structure and dynamic behavior of the denatured state, we have performed eleven 2.5 ns molecular dynamics simulations of reduced bovine pancreatic trypsin inhibitor (BPTI) at high temperature in water and a control simulation at 298 K, for a total of 30 ns of simulation time. In a neutral pH environment (acidic residues ionized), the unfolded protein structures were compact with an average radius of gyration 9% greater than the native state. The compact conformations resulted from the transient formation of non-native hydrophobic clusters, turns and salt bridges. However, when the acidic residues were protonated, the protein periodically expanded to a radius of gyration of 18 to 20 Å. The early steps in unfolding were similar in the different simulations until passing through the major transition state of unfolding. Afterwards, unfolding proceeded through one of two general pathways with respect to secondary structure: loss of the C-terminal helix followed by loss of β-structure or the opposite. To determine whether the protein preferentially sampled particular conformational substates in the denatured state, pairwise C α root-mean-square deviations were measured between all structures, but similar structures were found between only two trajectories. Yet, similar composite properties (secondary structure content, side-chain and water contacts, solvent accessible surface area, etc.) were observed for the structures that unfolded through different pathways. Somewhat surprisingly, the unfolded structures are in agreement with both past experiments suggesting that reduced BPTI is a random coil and more recent experiments providing evidence for non-random structure, demonstrating how ensembles of fluctuating structures can give rise to experimental observables that are seemingly at odds.

Titration Properties and Thermodynamics of the Transition State for Folding: Comparison of Two-state and Multi-state Folding Pathways

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (D physor I). CI2 folds from a largely unstructured denatured state under physiological conditions viaa transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (D phys→‡ ) is very similar for both proteins.

Identification and Characterization of the Unfolding Transition State of Chymotrypsin Inhibitor 2 by Molecular Dynamics Simulations

Temperature-induced unfolding of chymotrypsin inhibitor 2 (CI2) in water has been investigated using molecular dynamics simulations. One simulation (2.2 ns) has been analyzed in detail and three additional simulations (each≥1 ns) were performed to check the generality of the results. Concurrent loss of secondary and tertiary structure during unfolding was observed in all the simulations. For each simulation, the major transition state of unfolding was identified based on conformational analysis of protein structures along the unfolding trajectory. The transition state has a considerably weakened hydrophobic core and disrupted secondary structure. Nevertheless, the overall structure of the transition state is closer to the native state than to the unfolded state. The disruption of the hydrophobic core appears to be rate limiting. However, other energy barriers have to be overcome before reaching the major transition state. A method is described to quantitatively compare the structure of the simulated transition state with that characterized by protein engineering experiments. Good agreement with the experimental data is obtained for all four transition state models (the correlation coefficient R=0.80 to 0.93) and the average over all four models gives the best correlation ( R=0.94). These simulations provide the first comprehensive atomic-level view of what the unfolding transition state of C12 may look like.

Importance of two buried salt bridges in the stability and folding pathway of barnase.

The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition rate for unfolding, and a folding intermediate has been analyzed by protein engineering, kinetic, and thermodynamic studies. The aspartate residues in the bridges Arg69-Asp93 and Arg83-Asp75 were replaced by the isosteric analogue asparagine, while various replacements were probed for the positively charged arginine partners. The mutations are very destabilizing, lowering stability by up to 5.4 kcal/mol. A value of 3.0-3.5 kcal/mol was derived for the coupling energy between Arg and Asp from a double mutant cycle analysis. Despite the radical nature of these mutations, they do not appear to alter the pathway of folding. The interaction between Arg69 and Asp93, located in a relatively conserved region among ribonucleases, is predominantly formed in the major transition state along the folding pathway, as found previously from an analysis of more benign mutations; the value of phi(F) for all mutations at positions 69 and 93 are 0.8-0.9 in the major transition state for folding where phi(F) = 0 = fully unfolded and phi(F) = 1 = fully folded interaction energies). In contrast, the interaction between Arg83 and Asp75 in the active site of barnase is formed only in the native state of the protein. The analysis of folding pathways and the structure of folding intermediates by making kinetic and thermodynamic measurements on mutants appears even more robust than expected.

Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure. The structure of the transition state appears to be an expanded form of the native state in which secondary and tertiary elements have been partly formed concurrently. This is consistent with a "global collapse" model of folding rather than a framework model in which folding is initiated from fully preformed local secondary structural elements. This may be a general feature for the folding of proteins lacking a folding intermediate and is perhaps representative of the early stages of folding for multidomain or multimodule proteins. The major transition state for the folding of barnase, for example, has some fully formed secondary and tertiary structural elements in the major transition state, and barnase appears to form by a framework process. However, the fully formed framework may be preceded by a global collapse, and a unified folding scheme is presented.

Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.

Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics simulations. The major transition state of unfolding was identified on the basis of structural and conformational changes in the protein during the unfolding reaction. The native tertiary contacts in the hydrophobic core were considerably disrupted in the transition state, whereas the secondary structure was partially intact. The extent of structural change of the protein around a particular residue was represented quantitatively by the ratio of the number of contacts the residue makes in the transition state relative to the native state, phi MD, which allows quantitative comparison with the experimentally determined phi F values. For the region of the unfolding trajectory that is identified as the transition state, the phi MD and phi F values are in good agreement, suggesting that the transition state identified in the unfolding simulation corresponds to that probed with protein engineering methods. Although speculative, the transition state identified in the simulation is consistent with available experimental data and provides an in-depth view of what the transition state of unfolding may look like.

The folding of an enzyme: IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure

The pathway of refolding of barnase has been analysed by the protein engineering method using φ plots. The description comprises a folding intermediate, a major transition state (the unfolding transition state) and the fully folded structure. Over 40 mutations have been analysed in the different structural motifs, frequently with several probes in each region. Many of the mutations in this study give φ values for formation of the intermediate of 0, showing that the relevant regions of the structure are as fully unfolded in the intermediate as the unfolded state. Some folding φ values are close to unity, indicating that those regions are fully formed in the intermediate. Even if the data do not report back on a single intermediate but give the averaged properties of a heterogeneous population of sequential or parallel intermediates, then this simplicity of φ data shows that the intermediates tend to have structural features in common. Many φ values are intermediate between those for the unfolded state and the transition state, consistent with either partial structure formation in a single intermediate or a heterogeneous mixture of populations, although the former is more likely. The data are consistent with the intermediate, or collection of intermediates, being on the reaction pathway, rather than side products, because the φ values increase throughout the folding pathway. The main conclusions on the formation of substructure and sequence of folding events from the φ plots are as follows. (1) The major hydrophobic core (core 1) begins to form in the intermediate and strengthens in the major transition state. The centre of the core is formed earlier and is stronger in the intermediate and in the transition state than are the edges. (2) Core 2 is not formed until after the major transition state. (3) Core 3 begins to form in the intermediate and is compact in the transition state. (4) Loop 2, loop 4 and part of loop 1 do not fold until after the major transition state, but the guanosine-binding loop (loop 3) is formed in the intermediate and loop 5 is partially formed in the intermediate and the transition state. (5) The centre of the β-sheet is substantially formed in the intermediate, and is fully present in the transition state, but the edges, and associated turns, are definitely weakened. (6) The C-terminal two-thirds of helix 1 is substantially formed in the intermediate, but progressively unwinds from residues 8 to 6. (7) The N terminus of the protein and the N terminus of helix 2 are unfolded in the intermediate and transition state but the C terminus of the protein is substantially docked onto a β-strand and α-helix 1.

The folding of an enzyme: VI. The folding pathway of barnase: Comparison with theoretical models

The sequence of events in the refolding pathway of barnase has been analysed to search for general principles in protein folding. There appears to be a correlation between burying hydrophobic surface area and early folding events. All the regions that fold early interact extensively with the β-sheet. These interactions involve predominantly hydrophobic interactions and the burial of very extensive hydrophobic areas in which multiple, close, hydrophobic-hydrophobic contacts are established around a central group of aliphatic residues. There is no burial of hydrophilic residues in these regions; those that are partly screened from the solvent make hydrogen bonds. All the regions or interactions that are made late in the folding pathway do not make extensive contacts with the β-sheet. Their buried hydrophobic regions lack a central hydrophobic residue or residues around which other hydrophobic residues pack. Further, in some of these regions there is an extensive burial of hydrophilic residues. The results are consistent with one of the earlier events in protein folding being the local formation of native-like secondary structure elements driven by local hydrophobic surface burial. A possible candidate for an initiation site is a β-hairpin between β-strands 3 and 4 that is conserved in the microbial ribonuclease family. A comparison of structures in this family shows that those regions that can be superimposed, or have sequence homology, correspond to elements of structure that are formed and interact with each other early in the folding pathway, suggesting that some of these residues could be involved in directing the folding process. The data on barnase combined with results from other laboratories suggest the following tentative conclusions for the refolding of small monomeric proteins. (1) The refolding pathway is, at least in part, sequential and of compulsory order. (2) Secondary structure formation is driven by local hydrophobic surface burial and precedes the formation of most tertiary interactions. These elements are then stabilized and sometimes elongated by tertiary interactions. It is plausible that there are stop signals encoded in the linear sequence that prevent the elongation of isolated secondary structure elements in solution to a larger extent than is found in the folded protein. (3) Many tertiary interactions are not very constrained in the intermediate but become more and more defined as the hydrophobic cores consolidate, loop structures form and the configuration of surface residues takes place. The interactions between different elements of secondary structure are the last ones to be consolidated while the interactions within the secondary structure elements are consolidated earlier. It is possible that consolidation results partly from the expulsion of water molecules. (4) Tertiary interactions in the final major transition state are more constrained than in the intermediate, especially at the centre of the hydrophobic cores.