Transient 2D IR spectroscopy of ubiquitin unfolding dynamics

Abstract

Transient two-dimensional infrared (2D IR) spectroscopy is used as a probe of protein unfolding dynamics in a direct comparison of fast unfolding experiments with molecular dynamics simulations. In the experiments, the unfolding of ubiquitin is initiated by a laser temperature jump, and protein structural evolution from nanoseconds to milliseconds is probed using amide I 2D IR spectroscopy. The temperature jump prepares a subensemble near the unfolding transition state, leading to quasi-barrierless unfolding (the ''burst phase'') before the millisecond activated unfolding kinetics. The burst phase unfolding of ubiquitin is characterized by a loss of the coupling between vibrations of the ␤-sheet, a process that manifests itself in the 2D IR spectrum as a frequency blue-shift and intensity decrease of the diagonal and cross-peaks of the sheet's two IR active modes. As the sheet unfolds, increased fluctuations and solvent exposure of the ␤-sheet amide groups are also characterized by increases in homogeneous linewidth. Experimental spectra are compared with 2D IR spectra calculated from the time-evolving structures in a molecular dynamics simulation of ubiquitin unfolding. Unfolding is described as a sequential unfolding of strands in ubiquitin's ␤-sheet, using two collective coordinates of the sheet: (i) the native interstrand contacts between adjacent ␤-strands I and II and (ii) the remaining ␤-strand contacts within the sheet. The methods used illustrate the general principles by which 2D IR spectroscopy can be used for detailed dynamical comparisons of experiment and simulation. molecular dynamics ͉ protein folding ͉ temperature jump ͉ time-resolved spectroscopy F rom one perspective, protein folding is a chemical dynamics problem concerning description of the interplay of noncovalent interactions that involve the protein and surrounding solvent and exploration of the configurational space that leads to formation of the native structure. Although individually these interactions act on short time and distance scales, collectively they result in nanometer-scale conformational changes observed over time scales from picoseconds to seconds. The vast range of length and time scales, and the collective nature of folding coordinates, ensure that no single technique can time-resolve all relevant structural changes in solution (1-3). Most experimental methods favor either temporal or structural resolution and are limited to characterizing folding rates (kinetics) rather than mechanism (dynamics). From a computational approach, molecular dynamics (MD) simulations offer detailed dynamical information at atomic resolution for single molecules (4). Yet, simulation of folding is also challenged by the microsecond or longer time scales required, the need for extensive sampling of the ensemble, and only indirect experimental benchmarks. These challenges have spurred an interest in comparison between experiment and simulation that builds on their combined strengths to address mechanistic questions (5, 6).As an ultrafast vibrati...

Molecular dynamics with restrictions derived from optical spectra

“…7,8 Other important ways to increase effective resolution of the optical spectra include site-specific labeling with stable isotopes 9 and twodimensional multiphoton spectra sampling the chromophore coupling. 10 Simulations by modern quantum-chemical methods represent powerful tools for the spectra interpretations. 7,11,12 By comparison of the computed and experimental Raman and ROA spectral shapes not only the equilibrium structure but also an important knowledge about molecular flexibility and multiconformational states could be acquired.…”

Two-dimensional infrared spectra of isotopically diluted amyloid fibrils from Aβ40

“…Under these conditions a quantitative analysis of the spectra-structure relationships can be obtained. The 2D IR spectra of amyloid fibrils with 13 CA 18 O substitutions exhibit novel manifestations of the extended 3D structure of the fibrils, including the spatial arrangement of the coupled amide units and the presence of both static and fluctuating electric fields.…”

“…There has been a considerable amount of experimental work relating the 2D IR spectra of small peptide aggregates to their structure (13)(14)(15)(16)(17), and the recent experimental and theoretical applications of 2D IR have involved macromolecular systems such as folding proteins (18) and fibrils (19,20).…”

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

“…The 2DIR technique distinguishes β-sheet structures by producing Z shape like spectra [18,19] when this particular structural motif is present. In the past decades a number of 2DIR studies have been reported of peptides and proteins in solution [16,[20][21][22][23][24][25][26][27][28][29][30][31][32][33] or confined in membranes [34][35][36][37][38], revealing structural details and conformational changes from femtosecond (fs) to nanosecond (ns) time scales, and the nature of dynamic environments. For the structure determination of peptides that are in gas phase or micro-solvated (surrounded by few solvent molecules) the mid-infrared spectroscopic technique has become a promising tool [39][40][41][42].…”