C-D Bonds as Probes of Protein Motion

Background. One of the major obstacles to studying protein motion is the absence of probes with both high structural and temporal resolution. The most fundamental way to understand any molecule is to characterize the nature of the bonds that give the molecule its particular shape, stability, and flexibility (i.e. define the molecule’s potential energy surface). Of course, most chemical bonds may be characterized by IR spectroscopy. Thus, in principle any part of any protein could be characterized by IR spectroscopy. However, the inherent spectral congestion that results from the many overlapping IR absorptions in a protein has prevented use of the conventional spectroscopic methods employed to characterize small molecules.

Interestingly, all proteins have a ‘transparent window’ in their IR spectrum that is free of absorptions, between 1800–2700 cm-1. Any bond vibrations that absorb in this region are directly observable, even at high protein concentrations. Previous experiments have taken advantage of this window to observe small molecule heme ligands with suitable IR absorptions, such as carbon monoxide (~1950-2150 cm-1). In these studies, the absorption frequency and linewidth of the CO bond is used to characterize the ligand’s environment and dynamics; however, these studies do not characterize the protein itself. As a result, the protein is only probed indirectly.

A direct probe of proteins that also absorbs in the ‘transparent window’ of the protein IR spectrum is desirable for the study of protein motion. Consider, for example, the C–H bond, which has been a very useful probe of small molecules. It is predominantly local-mode in character, meaning that the absorption corresponds largely to the bond vibration, and is therefore easily interpreted. In principle then, the C–H bond would be an ideal probe of protein motion. The C–D bond possesses all of the spectroscopically desirable properties of a C–H bond, and has also been used as a probe of small molecules. Additionally, and most importantly, C–D bonds uniquely absorb within the transparent window, at ~2100 cm-1, are easily visualized when incorporated into a protein, and can be selectively incorporated at backbone or side chain positions through chemical protein synthesis or protein expression in defined media. In addition to providing high structural resolution, the C–D probe can also provide high time resolution. We are applying this methodology to a variety of different problems, including cytochrome c folding, ligand-receptor interactions, and motions important for catalysis in dihydrofolate reductase; click here to read more.

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