Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Abstract

Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfoldedstate ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Fö rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of Ϸ20 s at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of Ϸ2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.fluorescence resonance energy transfer ͉ guanidinium chloride ͉ protein folding ͉ RNase H ͉ single-molecule spectroscopy P roteins are complex systems that can exist in a huge number of different conformations. Even in the properly folded, native state, the polypeptide chain adopts many slightly different conformations that can be depicted as local minima in a rugged energy landscape, and relaxations and fluctuations in the landscape are crucially involved in functional processes (1, 2). Conformational heterogeneity is even more of a concern in studies of protein-folding reactions because of the vast number of possible arrangements of the unfolded polypeptide chain and the many complex pathways leading from the ensemble of unfolded to the native conformations in an overall funnel-shaped energy landscape (3-7).Because of Anfinsen's key observation that the native fold is already encoded in the sequence of amino acids (8), the proteinfolding problem has attracted enormous attention, and the field has progressed in a healthy interplay between theory and experiment. A wide variety of biophysical approaches were developed to follow the dynamics of the polypeptide en route to the native state. Valuable insights have been achieved by timeresolved spectroscopic experiments on bu...

Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

“…At more than 5 M GdmCl, the native peak was no longer obvious because of the low proportion of native state. The denatured peak shifted significantly to lower FRET efficiency with the increase of GdmCl concentration (from FRET efficiency of 0.65 at 3 M GdmCl to 0.49 at 6 M GdmCl), which was likely caused by the expansion of the denatured state at higher denaturant concentration, as observed in previous studies (6,21). There was a much less, but still observable, shift for the native peak between 0 and 2 M GdmCl (from 0.89 to 0.82 in FRET efficiency).…”

“…Direct observation of distinct states of BBL in the transition region at equilibrium would provide compelling evidence for barrier limited folding (6,13). The current method of choice is single-molecule fluorescence resonance energy transfer (SM-FRET) experiments, which has been widely applied in the studies of protein folding, structure, and function, and is especially useful in detection of heterogeneity of populations (14)(15)(16)(17)(18)(19)(20)(21).…”

“…We were not able to use SM-FRET to resolve unambiguously the folding mechanism of BBL because it folds in a fraction of a millisecond at room temperature, whereas the typical time resolution of SM-FRET was, until very recently, typically 1 ms (6,21,22), in which time BBL would rapidly equilibrate and appear as a time-averaged structure of the separate states if it folds by a barrier limited transition (23, 24). Here we study the folding of the protein by using improved equipment that allows study in the appropriate sub-millisecond time range.…”

Denaturant-induced Expansion and Compaction of a Multi-domain Protein: IgG

“…[11][12][13][14][15][16][17][18] In addition, several single-molecule fluorescence resonance energy transfer studies 26,29,32 have shown that conformational transitions within an unfolded protein ensemble could occur on a range of timescales, signifying the underlying hierarchical nature of the unfolded potential well. While these previous studies provided valuable insights into the statistical properties of unfolded polypeptide chains, they mainly focused on relatively small and mostly single-domain proteins.…”

Simulation of FRET dyes allows quantitative comparison against experimental data

“…16 Similarly, the specific dye shape and mutual orientation are crucial and are not fully captured by simple accessible volume calculations. 17 Molecular dynamics simulations also allow testing assumptions such as approximating the mutual dye-dye orientation factor κ 2 with 2/3, which is questioned in several studies, 18,19 in particular, for low linker flexibilities. 20 Many simulation protocols have computationally prohibitive demands to reach sufficiently long simulation times for accurate calculations of experimentally accessible properties.…”