Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition

Abstract

Proteins exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires knowledge of the populated states and thus the experimental tools to characterize them. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution, and 2D IR methods that provide richer information are becoming more routine. Although application of IR spectroscopy for investigation of proteins is challenged by spectral congestion, the issue can be overcome by site-specific introduction of amino acid side chains that have IR probe groups with frequency-resolved absorptions, which furthermore enables selective characterization of different locations in proteins. Here, we briefly introduce the biophysical methods and summarize the current progress toward the study of proteins. We then describe our efforts to apply site-specific 1D and 2D IR spectroscopy toward elucidation of protein conformations and dynamics to investigate their involvement in protein molecular recognition, in particular mediated by dynamic complexes: plastocyanin and its binding partner cytochrome f, cytochrome P450s and substrates or redox partners, and Src homology 3 domains and proline-rich peptide motifs. We highlight the advantages of frequency-resolved probes to characterize specific, local sites in proteins and uncover variation among different locations, as well as the advantage of the fast time scale of IR spectroscopy to detect rapidly interconverting states. In addition, we illustrate the greater insight provided by 2D methods and discuss potential routes for further advancement of the field of biomolecular 2D IR spectroscopy.

Lessons from combined experimental and theoretical examination of the FTIR and 2D-IR spectroelectrochemistry of the amide I region of cytochrome c

“…With future improvement of experimental and simulation procedures, a simultaneous fitting based on the FTIR as well as 2D-IR spectra may be within reach. However, it may be more fruitful to employ isotope labelling methods 17,18,[62][63][64][65][66][67][68][69] or IR labels [70][71][72][73][74][75][76][77][78] to reveal information on local structural changes.…”

Probing Ligand Effects on the Ultrafast Dynamics of Copper Complexes via Midinfrared Pump–Probe and 2DIR Spectroscopies

“…The structure–function relationship can also be established through a dynamical perspective. The interplay between ultrafast dynamics and catalytic activity has been elucidated in metalloenzymes − and their mimics, , as well as catalytic transition-metal complexes, − demonstrating that the characterization of ultrafast dynamics can foster a better understanding of the reaction mechanism and its relationship with molecular structures.…”

“…Two-dimensional infrared (2DIR) spectroscopy has proven to be a powerful tool to probe ultrafast equilibrium dynamics of enzymes as well as models of their active sites on femtosecond to picosecond timescales. − ,− Polarization-dependent mid-IR pump–probe spectroscopy has also been widely used to extract the vibrational population and orientational relaxation dynamics of various systems. − In this manuscript, we apply both polarization-dependent mid-IR pump–probe spectroscopy and 2DIR spectroscopy to a series of copper coordination complexes to determine how the structural variation in the secondary coordination sphere impacts the ultrafast dynamics at a small-molecule binding site where a series of trigonal pyramidal Cu I complexes bind O 2 .…”

Least-Squares Fitting of Multidimensional Spectra to Kubo Line-Shape Models

“…Some of these approaches include the nodal-line slope, − dynamic line width, ellipticity, , covariance, spectral phase slope, inhomogeneity index, eccentricity, centerline slope (CLS), inverse centerline slope (invCLS), , and the correlation coefficient obtained by a 2D Gaussian fit . The most popular among these has been the CLS method, which has been used to measure spectral diffusion in a wide variety of systems, including proteins, − lipid membranes, − and ionic liquids. − Relative to some other metrics, the CLS is more reliable at lower SNR, is invariant to line-shape interference from anharmonic peaks or phase twist, and is invariant to apodization time for time constants and relative amplitudes …”