New structural insights from Raman spectroscopy of proteins and their assemblies

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Abstract
Protein structure and stability are sensitive to and dependent on the local interactions of amino acid side chains. A diverse and important type of side-chain interaction is the hydrogen bond. Although numerous hydrogen bonds are resolved in protein 3-dimensional structures, those of the cysteine sulfhydryl group (S H) are elusive to high-resolution X-ray and NMR methods. However, the nature and strength of sulfhydryl hydrogen bonds (SH…X) are amenable to investigation by Raman spectroscopy. The power of the Raman method for characterizing SH…X interactions is illustrated by resolving the Raman SH stretching band for each of the eight cysteines per 666-residue subunit in the trimeric tailspike of icosahedral bacteriophage P22. The Raman sulfhydryl signatures of the wild-type tailspike and eight single-site cysteine to serine mutants reveal a heretofore unrecognized diversity of SH hydrogen bonds in a native protein. The use of Raman spectroscopy to identify the non-hydrogen-bonded state of the tyrosine phenoxyl group is also described. This unusual and unexpected state occurs for all tyrosines in the assembled capsids of filamentous viruses Ff and Pf1. The Raman spectral signature of the non-hydrogen-bonded tyrosine phenoxyl, which is characterized by an extraordinary Raman Fermi doublet intensity ratio (I850/I830 = 6.7), extends and refines the existing correlation for hydrogen-bonded tyrosines. Finally, a novel Raman signature for tryptophan in the Pf3 filamentous virus is identified, which is proposed as diagnostic of “cation–π interaction” involving the guanidinium group of Arg 37 as a cation donor and the indolyl ring of Trp 38 as a π-electron acceptor. These studies demonstrate the power of Raman spectroscopy for investigating the interactions of key side chains in native protein assemblies. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 214–225, 2002