Peptide Conformational Dynamics and Vibrational Stark Effects Following Photoinitiated Disulfide Cleavage

163

196

Ultrafast IR spectroscopy is used to monitor the nonequilibrium backbone dynamics of a cyclic peptide in the amide I vibrational range with picosecond time resolution. A conformational change is induced by means of a photoswitch integrated into the peptide backbone. Although the main conformational change of the backbone is completed after only 20 ps, the subsequent equilibration in the new region of conformational space continues for times >16 ns. Relaxation and equilibration processes of the peptide backbone occur on a discrete hierarchy of time scales. Albeit possessing only a few conformational degrees of freedom compared with a protein, the peptide behaves highly nontrivially and provides insights into the complexity of fast protein folding.

Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Picosecond conformational transition and equilibration of a cyclic peptide

Bredenbeck

Helbing

Sieg

et al. 2003

163

196

“…6A and supporting information). On such a surface, the protein does not have to fold through a single route (31)(32)(33)(34), resulting in a spread of time scales (35)(36)(37)(38). Fig.…”

Section: Discussionmentioning

confidence: 99%

Kinetics are probe-dependent during downhill folding of an engineered λ _6–85 protein

Gruebele

2005

100

140

The Y22W͞Q33Y͞G46,48A mutant of the protein 6 -85 folds in a few microseconds at room temperature. We find that its folding kinetics are probe-dependent under a strong bias toward the native state, a new signature for downhill folding. The IR-and fluorescence-detected relaxation time scales converge when the native bias is removed by raising the temperature, recovering activated two-state folding. Langevin dynamics simulations on one-and 2D free energy surfaces tunable from two-state to downhill folding reproduce the difference between the IR and fluorescence experiments, as well as the temperature and viscosity trends. In addition, the 2D surface reproduces the stretched exponential dynamics that we fit to the glucose solution experimental data at short times. Nonexponential dynamics at <10 s is a signature either for local free energy minima along the reaction coordinate (''longitudinal roughness''), or for folding on a higherdimensional free energy surface (''transverse roughness'').fluorescence ͉ infrared ͉ helix bundle ͉ amide band ͉ landscape roughness T he possibility of downhill (type 0) folding is one of the central but least expected predictions of the energy landscape theory. As discussed by Bryngelson et al. (1), folding could proceed downhill in free energy when there is a sufficiently strong bias toward the native state. When the bias is reduced by heat denaturation, activated folding over a barrier (the type 1 scenario) is recovered (1). This switch corresponds to a transition from potentially nonexponential diffusion on a rough free energy surface to activated kinetics with a single rate coefficient, k a . Type 1 folding seems to be the norm among natural proteins, but type 0 folding remains a possibility for proteins with an unusually strong native bias.Significant experimental progress has been made in the search for downhill folding kinetics (2). With the advent of fast laser initiation techniques (3), including laser T-jumps (4-6), the necessary nano-to microsecond time scales have become accessible. In 1999, Sabelko et al. (7) reported downhill formation of a phosphoglycerate kinase (PGK) folding intermediate. The nonexponential dynamics could be tuned toward a single exponential by cold or heat denaturing the protein, decreasing the bias toward the native state. The C-terminal domain of PGK was later shown to be responsible for this behavior (8). Kinetic evidence indicating downhill folding to the native state has also been reported. Activated kinetics of the engineered 6-85 protein were preceded by a fast (k m Ϸ 1 s Ϫ1 ) ''molecular phase'' (9). This new phase was attributed to a substantial population en route between the native and denatured states and accounted for nearly the entire signal in stabilizing glucose solution (10). The experimental downhill folding ''speed limit'' is in agreement with the preexponential factor calculated by Portman et al. (11). Estimates based on diffusion of denatured proteins (12, 13), on generic folding models (14), and on molecular dynamics studies of th...

“…2) (22,23). This method observes the opposite of loop formation, namely the separation of two residues that initially are in close contact.…”

mentioning

confidence: 99%

Measurement of energy landscape roughness of folded and unfolded proteins

Milanesi

Waltho

Hunter

et al. 2012

Self Cite

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k B T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k B T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. photochemical trigger | subdiffusion M ajor advances have been made in recent years in understanding dynamic aspects of protein conformational changes, particularly protein folding; however, many issues remain to be solved (1). Among these are the properties of the unfolded protein ensemble and the role of residual structure of denatured proteins in promoting folding (2), the heterogeneity of microscopic folding pathways (3), and the existence of multiple distinct, but only transiently populated, intermediates (4). Particularly for fast-folding proteins, the idea of downhill folding, i.e., the absence of a significant barrier, has been suggested as an alternative mechanism (5, 6), but it is not clear to what extent fast-folding proteins make use of this mechanism. On the other hand, technical progress has made it possible to observe multiple folding and unfolding events in millisecond all-atom molecular dynamics simulations. Such simulations have shown that some proteins always follow the same folding pathway, whereas others have several different pathways (7). Moreover, individual folding events occur with submicrosecond transit times through a distinct transition state but are separated by long waiting times, which yield the experimentally observed folding times (8).The idea of motion on a rugged energy landscape (9-11) has been used widely to describe conformational changes in protei...