Infrared Studies of Fast Events in Protein Folding

157

187

Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with nonlinear IR spectroscopy of the amide I vibrations. Equilibrium temperaturedependent 2D IR spectroscopy reveals the unfolding of the ␤-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from delocalized vibrations of the ␤-sheet. Transient unfolding after a nanosecond temperature jump is monitored with dispersed vibrational echo spectroscopy, a projection of the 2D IR spectrum. Whereas the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans 6 decades in time. The transient behavior can be separated into fast and slow time scales. From 100 ns to 0.5 ms, the spectral features associated with ␤-sheet unfolding relax in a sequential, nonexponential manner, with time constants of 3 s and 80 s. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands III-V of the ␤-sheet before unfolding of the hairpin that forms part of the hydrophobic core. This downhill unfolding is followed by exponential barrier-crossing kinetics on a 3-ms time scale.protein-folding dynamics ͉ temperature jump ͉ nonlinear IR spectroscopy D escribing the conformational changes of proteins as they fold from a disordered denatured state to a compact native state remains an important experimental objective. Studies that examine this subject provide a molecular interpretation to the conceptual framework of the energy landscape picture (1-3) and allow more direct comparison of experiment and simulation (4, 5). Viewed as a problem in molecular dynamics, characterizing protein folding poses considerable challenges because it calls for a statistical yet structurally sensitive description of a heterogeneous ensemble in solution evolving over many decades in time.Most protein-folding experiments measure kinetics: the rate of appearance or disappearance of an experimental signature for a particular species, typically on millisecond or longer time scales. These results give information on the height of energetic barriers much higher than thermal energy but say little about how structure changed along the path. A number of fast folding experiments of proteins and peptides have shown that downhill folding, in which evolution is governed by energy barriers ՇkT, can be initiated with a nanosecond temperature jump (T-jump). Such experiments work in a diffusive regime that allows a freer exploration of available structures and provide evidence that the relevant molecular time scales for folding is nanoseconds to microseconds (6-9). If downhill folding can be initiated and followed with a structure-sensitive probe, then meaningful information can be obtained on the underlying molecular dynamics of folding. We report here on such an experiment, a conformationally sensitive probing of downhill unfolding of ubiquitin over nanosecond-to-millise...

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confidence: 99%

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confidence: 99%

Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin

Chung

Khalil

Smith

et al. 2005

157

187

“…Essentially all folding rate constants in the 10 4 to 10 7 ͞s range have been derived from fluorescence-or IR-monitored temperature jump experiments (12,13,18,19). Two groups have recognized the potential of NMR spectroscopy to probe 10-to 50-s exchange phenomena (20-22).…”

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confidence: 99%

Hairpin folding rates reflect mutations within and remote from the turn region

Olsen

Fesinmeyer

Stewart

et al. 2005

149

Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin (k F 278 ‫؍‬ 1.5 ؋ 10 4 ͞s). GB1 hairpin analogs spanning a wide range of thermodynamic stabilities (⌬G U 298 ‫؍‬ ؊3.09 to ؉3.25 kJ͞mol) were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive sidechain͞side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by ''zipper'' folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.␤-hairpin ͉ exchange broadening ͉ folding dynamics ͉ loop search P rotein engineering experiments indicate that ␤-hairpins appear as transition-state features in numerous folding pathways. Hairpin redesign has resulted in changes in protein-folding mechanisms (1), and hairpin stabilization can accelerate (1-3) or retard (2, 4) protein folding. The protein-folding problem continues to be of high interest for at least three reasons: predicting structures from genome-derived sequences, improving a priori protein-fold design, and enhancing our understanding of the mechanisms of protein misfolding diseases (6, 7). Folding rates have been of particular interest, with more examples of redesigned proteins that fold near the calculated protein-folding speed limit (8) appearing regularly. For ␤-sheet proteins and ␤ oligomers (as found in amyloid fibrils formed from misfolded protein states), hairpin dynamics play a key role.Although there is an increasing body of data on hairpin folding dynamics (9-15), with one exception these have not included a set of probing mutations to address specific questions. That exception (14) suggests that loops with a greater turn preference accelerate folding to a much greater extent than the optimization of hydrophobic interactions in the folded state. Other data (12) suggest that the length of the loop connecting the hydrophobic residues that form hairpin-stabilizing cross-strand interactions is reflected in the folding rate. Throughout, hairpin folding has been modeled as a two-state equilibrium. Whether hairpin͞coil transitions are 1-s versus 50-s events has significant consequences. Peptide helix nucleation is a sub-s event (10,16,17), which allows helix formation to be a preequilibrium event relative to the hydrophobic-collapse stage o...

“…In the last decade, laser T-jump has emerged as a powerful tool whereby conformational changes in biomolecules can be monitored on time scales of submicroseconds-tomilliseconds (23)(24)(25)(26)(27)(28)(29). The T-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA (Fig.…”

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confidence: 99%

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Kuznetsov¹,

Sugimura

Vivas³

et al. 2006

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNAbinding sites. Previous kinetics measurements on several DNAbending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending͞unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from Ϸ2 ms ؊1 at Ϸ37°C to Ϸ40 ms ؊1 at Ϸ10°C and correspond to an activation energy of Ϸ14 ؎ 3 kcal͞mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend͞kink DNA before forming a tight complex with IHF.DNA bending kinetics ͉ laser temperature jump ͉ protein-DNA interactions ͉ time-resolved FRET measurements