Proteins in solution can take on multiple conformational states that are vital to function. However, study of the conformational states of proteins is impeded by spatial heterogeneity of proteins and the timescale on which they interconvert, which occur over a range from picoseconds to milliseconds. Infrared (IR) spectroscopy is a powerful technique that has high temporal resolution needed to observe these wide variety of timescales and bond-specific spatial resolution. The use of IR to study proteins has been limited due to spectral congestion of proteins arising from the large number of similarly bonded atoms. Fortunately, there exists within protein spectra a ‘transparent’ frequency window (1800 –2500 cm-1). A vibrational probe having an absorption within this frequency window can be site-selectively incorporated into proteins that is sensitive to its local environment. Carbon-deuterium (C-D) bonds are ideal probes due to their non-perturbative nature in proteins. This work focuses on use of d3-L-proline (d3Pro) as a vibrational probe of molecular recognition of three peptides by a Src Homology 3 (SH3) domain. SH3 domains recognize the motif PxxP, so d3Pro is a valuable probe of the recognition sequence. The asymmetric stretch of the CδD2 group of the incorporated d3Pro shows sensitivity to the local conformation, specifically to proline-ring pucker states. Changes in this vibration when the peptides bind to AbpSH3 indicate variation in mechanisms of molecular recognition by the SH3 domain.

Elucidating conformational heterogeneity involved in proline-rich motif recognition via infrared spectroscopy

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