Raman Analysis of Cysteine Thiols in Proteins

Stephen W. Raso
(in collaboration with Prof. George J. Thomas, Jr., University of Missouri-Kansas City)

Many proteins contain cysteine residues which are not involved in active sites, ligand binding or disulfide bonds. S-H stretching vibrations generate characteristic Raman bands, which are highly dependent on the environment of the sulfhydryl proton, and represent a valuable method for examining S-H interactions under physiological conditions (1). For the trimeric tailspike protein from bacteriophage P22, all eight cysteine residues (per monomer) are reduced in the native trimer, yet a subset facilitates proper subunit folding and assembly (2). Raman spectroscopy of the tailspike resolves the S-H region of the spectrum into multiple bands, indicating sulfhydryls in several environments (Fig. 1).

Some of the tailspike S-H bands are at extremely low wavenumbers; the band corresponding to one S-H vibration is at 2530 cm-1, a frequency lower than any reported previously. The complex band pattern in this region is diagnostic of the native folded tailspike protein. The Raman spectra of eight Cys to Ser mutant proteins have been analyzed permitting the identification of the specific contribution from each cysteine sulfhydryl to the various S-H bands (Fig. 2).

In addition, the hydrogen bonding environment of each of the eight tailspike cysteines was elucidated (Table 1). Raman spectroscopy provides H-bonding information about the native solution-state of a protein, unlike X-ray crystallographic methods wherein H-bonding interactions are inferred by the interatomic distances of potential H-bond donors and acceptors in the crystalline state.

Table 1: H-bonding Environment of Each Tailspike Cysteine Thiol

Cysteine Residue	Raman band (cm-1)	Proposed H-bonding Strength
169	2565	Moderately Strong
267	2550 (90%) 2585 (10%)	Strong Weak/Acceptor
287	2550 (63%) 2585 (37%)	Strong Weak/Acceptor
290	2585	Weak/Acceptor
458	2550	Strong
496	2585	Weak/Acceptor
a613	2530	Very Strong
635	2576	Moderately Weak

^aThe 2530 cm-1 Raman marker of Cys 613 represents the strongest S-H--X hydrogen bond observed for a cysteine or model thiol in solution.

These techniques can be applied to other proteins to study the specific roles cysteine residues in protein structure and function. In addition, S-H Raman markers may be used to probe changes in protein conformation and cysteine oxidation.

References

1. Li, H. & Thomas, G.J., Jr. (1991) J. Am. Chem. Soc. 113, 456-462.

2. Robinson, A.S. & King, J. (1997) Nature Struct. Biol. 4, 450-455.