<p> Conformational heterogeneity and protein dynamics play important roles in molecular recognition, but are experimentally difficult to characterize with sufficient temporal and spatial resolution. Infrared (IR) spectroscopy can probe protein dynamics on sub-ps timescales; and further, the small size of vibrational chromophores combined with site-selective incorporation of spectrally isolated IR probes provides high spatial resolution. Herein, we site-specifically introduce nitrile and carbon-deuterium bonds at distinct sites in the Src-homology 3 (SH3) domain from yeast protein Sho1 and its proline-rich peptide binding partner from Pbs2 to examine the underlying mechanisms of molecular recognition via IR spectroscopy. Further, we present efforts at developing instrumentation aimed at improving characterization of weakly absorbing vibrational probes in strongly absorbing solvent.</p><p> Nitrile probes were introduced at six distinct sites in the SH3 domain via amber codon suppression. Variation between the observed absorbance bands indicates site specific differences in conformational heterogeneity imposed by protein domain. Residue-specific changes upon peptide binding are observed at incorporated nitrile moieties, but are more dramatically observed for deuterated vibrational probes incorporated within the peptide binding partner. Deuterated amino acids were incorporated at highly conserved proline residues within the peptide ligand. Upon binding, absorbance bands are observed which indicate population of multiple conformational states in the bound complex. Only single resonances were observed by characterization of the same labeled bonds by NMR, suggesting rapid interconversion on the NMR timescale. Results suggest that the SH3 domain recognizes its peptide binding partner with at least elements of an induced-fit mechanism.</p><p> Characterization of the vibrational probes used above can be challenging due to the path length limitation imposed by the presence of strongly absorbing solvent water. This places an upper bound on the achievable signal strength which can obscure small (µOD) absorbance bands. To confront this limitation we have constructed an absorbance spectrometer with a quantum cascade laser (QCL) source. The instrument allows characterization of samples of increased path length with similar signal-to-noise ratios as in FT IR measurements. Achievable signal-to-noise ratios are limited by QCL source noise; we present several approaches, one electronic and one interferometric, aimed at limiting the deleterious effect of QCL fluctuations.</p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10977701 |
| Date | 28 November 2018 |
| Creators | Horness, Rachel E. |
| Publisher | Indiana University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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