Site-specific 2D IR spectroscopy: a general approach for the characterization of protein dynamics with high spatial and temporal resolution

Abstract

The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p-cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function.

Fig. 1

Structural model of the complex of SH3^Sho1 and pPbs2 (2VKN) showing locations of CNF incorporation.

Fig. 2

Example 2D IR spectra of CNF8 (left panel), CNF10 (center panel), and CNF54 (right panel) SH3^Sho1 at T_w of 0.25 and 1 ps for the unligated protein (top row) and the pPbs2 complex (bottom row). Overlayed are the center lines from analysis of the lineshapes of the average spectra (white dotted line).

Fig. 3

CLS decay curves (points) and fits (lines) for unligated SH3^Sho1 (colored) and the pPbs2 complex (black).

Fig. 4

Binding-induced changes in the timescale of dynamics (upper panel) and the inhomogeneous distribution of frequencies sampled rapidly (middle panel) and slowly (bottom panel) reflected by the FFCFs. Error bars are standard deviations from three sets of experiments with independently prepared samples.

Fig. 5

RDFs for distance of the cyano nitrogen to (A) water oxygen atoms, (B) all heavy atoms excluding solvent and CNF side chain, and (C) change in the RDFs for these heavy atoms upon ligand binding for CNF2 (orange), CNF8 (teal), CNF10 (blue), CNF16 (red), CNF20 (green), and CNF54 (purple) SH3^Sho1.

Fig. 6

Overlay of average structures from MD simulations showing side chains of residues in local environment of (A) CNF10 and (B) CNF54 when unligated (green) and bound to pPbs2 (teal).