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Every residue on a protein can be characterized by its interaction with water, in lack or in excess, as water is the matrix of biological systems. Infrared spectroscopy and the implementation of local azidohomoalanine (AHA) probes allow us to move beyond an ensemble or surface-driven conceptualization of water behavior and toward a granular, site-specific picture. In this paper, we examined the role of crowding in modulating both global and local behavior on the β-hairpin, TrpZip2 using a combination of Fourier-transform infrared spectroscopy (FTIR) spectroscopy, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics simulations. We found that, at the amino acid level, crowding drove dehydration of both sheet and turn peptide sites as well as free AHA. However, the subpicosecond dynamics showed highly individualized responses based on the local environment. Interestingly, while steady-state FTIR measurements revealed similar responses at the amino-acid level to hard versus soft crowding (dehydration), we found that PEG and glucose had opposite stabilizing and destabilizing effects on the protein secondary structure, emphasizing an important distinction in understanding the impact of crowding on protein structure as well as the role of crowding across length scales. 

Thiocyanates, nitriles, and azides represent a versatile set of vibrational probes to measure the structure and dynamics in biological systems. The probes are minimally perturbative, the nitrile stretching mode appears in an otherwise uncongested spectral region, and the spectra report on the local environment around the probe. Nitrile frequencies and lineshapes, however, are difficult to interpret, and theoretical models that connect local environments with vibrational frequencies are often necessary. However, the development of both more accurate and intuitive models remains a challenge for the community. The present work provides an experimentally consistent collection of experimental measurements, including IR absorption and ultrafast two-dimensional infrared (2D IR) spectra, to serve as a benchmark in the development of future models. Specifically, we catalog spectra of the nitrile stretching mode of methyl thiocyanate (MeSCN) in fourteen different solvents, including non-polar, polar, and protic solvents. Absorption spectra indicate that π-interactions may be responsible for the line shape differences observed between aromatic and aliphatic alcohols. We also demonstrate that a recent Kamlet–Taft formulation describes the center frequency MeSCN. Furthermore, we report cryogenic infrared spectra that may lead to insights into the peak asymmetry in aprotic solvents. 2D IR spectra measured in protic solvents serve to connect hydrogen bonding with static inhomogeneity. We expect that these insights, along with the publicly available dataset, will be useful to continue advancing future models capable of quantitatively describing the relation between local environments, line shapes, and dynamics in nitrile probes.