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.

Site-Selective Dynamics of Ligand-Free and Ligand-Bound Azidolysozyme

“…11,12 Isotope edited carbonyl spectroscopy was used to characterize the mechanism of protein folding and amyloid formation 13,14 or the structure and function of membrane proteins. 15,16 Additional molecular groups such as thiocyanate, 17 cyanamide, 18 sulfhydryl vibrations of cysteines, 19 deuterated carbons, 20 carbonyl vibrations of metal-carbonyls, 21 cyanophenylalanine, 22 and azidohomoalanine (Aha) 23 have also been explored.…”

“…Such an approach relies on the sensitivity of the probe to report on changes in the vibrational frequencies induced by alterations in the local electrostatic interactions in the vicinity of the probe. 22 IR spectroscopy is a potentially advantageous technique to characterize ligand binding to proteins. 31,32 Its success depends in part on the notion that when a ligand binds to a protein, the frequency of an infrared active vibration shifts due to the different electric field in solution -often water -and in the protein binding site.…”

“…For positions Ala63, Ala73 and Ala160 the differences are 8, 3, and 2 cm −1 , respectively, whereas for the other residues the change is within 1 cm −1 . Such frequency changes can be measured with stat-of-the art experiments 22 and their magnitude is also consistent with previous simulations of the vibrational Stark effect for the -CN probe in PhCN with red shifts of up to 3.5 cm −1 in going from the WT to the L99A and L99G mutants of T4-Lysozyme 32 Similarly, the 1D and 2D infrared spectroscopy of -CO as the label for insulin monomer and dimer found 45 that the relative shifts of the spectroscopic response was correctly described whereas the absolute frequencies may differ by some 10 cm −1 . In a very recent work such an approach found a splitting of 13 cm −1 , compared with 25 cm −1 from experiment, for the outer and central -CO labels in cationic trialanine in water.…”

“…49 Thus, differences of ∼ 1 cm −1 for the frequency of the reporter in different chemical environments can be experimentally detected. 22 From the frequency trajectories the frequency fluctuation correlation function (FFCF) can be determined which contains valuable information on relaxation time scales corresponding to the solvent dynamics around the solute. The FFCFs are fit to an empirical expression…”

“…Changes in the maximum of the infrared absorbance are of the order of one to several cm −1 which is still detectable with state-of-the-art experiments. 22 Given that the -N 3 label can be introduced at multiple positions along the polypeptide chain with specific spectroscopic signatures for each variant of the system, it may even be possible to use the present approach to refine existing structural models based on NMR measurements 58 or from more conventional co-crystallization and X-ray structure determination efforts.…”