Barrierless Folding Research Articles

How the Barrierless Folding Helps DNA Recognition: Theoretical Investigation on Ultrafast Folding Protein Engrailed Homeodomain

Engrailed homeodomain (EngHD), a 3-helix bundle fast-folding protein, has been found to play a critical role in transcription regulation during its binding to DNA. Evidence shows that protein efficiently recognizes the short target DNA sequence from the enormous pool of binding sites via the process of “facilitated diffusion”, including sliding and hopping. During the facilitated diffusion, protein is supposed to go through “speed” mode, where protein nonspecifically interacts with DNA in fast diffusion, and “stability” mode, where protein strongly and specifically scans the base pairs in DNA with slow displacement.

Analyzing Protein Folding Cooperativity by Differential Scanning Calorimetry and NMR Spectroscopy

Some marginally stable proteins undergo microsecond time scale folding reactions that involve significant populations of partly ordered forms, making it difficult to discern individual steps in their folding pathways. It has been suggested that many of these proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) relaxation dispersion experiments, and an analysis of the temperature dependence of amide (1)H and (15)N chemical shifts. We applied this method to the PBX homeodomain (PBX-HD), which folds on the microsecond time scale and produces a broad DSC thermogram with an elevated and steeply sloping native-state heat capacity baseline, making it a candidate for barrierless folding. However, by globally fitting the NMR thermal melt and DSC data, and by comparing these results to those obtained from the NMR relaxation dispersion experiments, we show that the native form of the protein undergoes two-state exchange with a small population of the thermally denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms and firmly establishes that folding of PBX-HD is cooperative. Further, we see evidence of large-scale structural and dynamical changes within the native state by NMR, which helps to explain the broad and shallow DSC profile. This study illustrates the potential of combining calorimetry with NMR dynamics experiments to dissect mechanisms of protein folding.

Direct Observation of Downhill Folding of λ-Repressor in a Microfluidic Mixer

The protein λ 6-85 has been implicated in barrierless folding by observations of kinetic relaxation after nanosecond T-jump. In this work we observed folding of this protein after dilution of a high denaturant in an ultrarapid microfluidic mixer at temperatures far below the thermal midpoint. The observations of total intensity and spectral shift of tryptophan fluorescence yielded distinctly different kinetics and activation energies. These results may be explained as diffusion on a low-barrier, one-dimensional, free-energy surface, with different probes having different sensitivities along the reaction coordinate. Additionally, we observed an extremely fast phase within the mixing time that was not observed by T-jump, suggesting that the ensemble of unfolded states populated at high denaturant is distinct from those accessible at high temperature.

Understanding protein folding cooperativity based on topological consideration

The folding cooperativity is an important issue of protein folding dynamics. Since the native topology plays a significant role in determining the folding behavior of proteins, we believe that it also has close relationship with the folding cooperativity. In the present work, we perform simulations on proteins Naf-BBL, QNND-BBL, CI2, and SH3 with the Gō model and compare their different folding behaviors. By analyzing the weak cooperative folding of protein Naf-BBL in detail, we found that the folding of Naf-BBL shows relatively weak thermodynamic coupling between residues, and such weak coupling is found mainly between the nonlocal native contacts. This finding complements our understandings on the source of barrierless folding of Naf-BBL and promotes us to analyze the topological origins of the poor thermodynamic coupling of Naf-BBL. Then, we further extend our analysis to other two-state and multistate proteins. Based on the considerations of the thermodynamic coupling and kinetic coupling, we conclude that the fraction of scattered native contacts, the difference in loop entropy of contacts, and the long range relative contact order are the major topological factors that influence the folding cooperativity. The combination of these three tertiary structural features shows significant correlations with the folding types of proteins. Moreover, we also discuss the topological factors related to downhill folding. Finally, the generic role of tertiary structure in determining the folding cooperativity is summarized.

Cold destabilization and temperature jump of the murine prion protein mPrP(23–231)

We analyzed the thermal stability of the recombinant murine prion protein mPrP(23–231) with a single tryptophan mutation (F174W) and its perturbation by cold temperature. Compared to the N-terminally truncated ones, full-length construct is significantly unstable and forms intermediate state of urea denaturation, and also undergoes the cold destabilization under the ambient pressure. In order to detect the very early phase of the folding, we also applied a laser-induced temperature jump kinetic measurement and observed a kinetic phase of several microseconds, suggesting the barrierless folding process. The conformational instability and low barriers between different conformers may explain the unusual flexibility leading to the pathogenic conversion and the strain diversity.

Origins of barriers and barrierless folding in BBL

In contrast to classical chemical phenomenology, theory suggests that proteins may undergo downhill folding without an activation barrier under certain thermodynamic conditions. Recently, the BBL protein was proposed to fold by such a downhill scenario, but discrepancies between experimental results found in different groups argue against this. After briefly reviewing the major experimental studies of the BBL folding mechanism, we show that simulations of both coarse-grained and atomistic models can reconcile the seemingly conflicting observations.

Backbone dynamics, fast folding, and secondary structure formation in helical proteins and peptides

We discuss the construction of a simple, off-lattice model protein with a comparatively detailed representation of the protein backbone, and use it to address some general aspects of the folding kinetics of a small helical protein and two peptide fragments. The model makes use of an associative memory hamiltonian to smoothly interpolate between the limits of a native contact only, or Go, potential and a statistical pair potential derived from a database of known structures. We have observed qualitatively different behavior in these two limits. In the Go limit, we see apparently barrier-less folding. As we increase the roughness of the model energy landscape, we can observe the emergence of the characteristic activated temperature dependence previously seen in lattice studies and analytical theories. We are also able to study the dependence of the folding kinetics on local interactions such as hydrogen bonds, and we discuss the implications of these results for the formation of secondary structure at intermediate stages of the folding reaction.

Backbone dynamics, fast folding, and secondary structure formation in helical proteins and peptides

We discuss the construction of a simple, off-lattice model protein with a comparatively detailed representation of the protein backbone, and use it to address some general aspects of the folding kinetics of a small helical protein and two peptide fragments. The model makes use of an associative memory hamiltonian to smoothly interpolate between the limits of a native contact only, or Gō, potential and a statistical pair potential derived from a database of known structures. We have observed qualitatively different behavior in these two limits. In the Gō limit, we see apparently barrier-less folding. As we increase the roughness of the model energy landscape, we can observe the emergence of the characteristic activated temperature dependence previously seen in lattice studies and analytical theories. We are also able to study the dependence of the folding kinetics on local interactions such as hydrogen bonds, and we discuss the implications of these results for the formation of secondary structure at intermediate stages of the folding reaction. Proteins 1999;34:281–294. © 1999 Wiley-Liss, Inc.