A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy

We have developed a holistic protein structure estimation technique using a mide I band Raman spectroscopy. This technique combines the superposition o f reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applica ble to solution, suspension, and solid protein samples, A key component of this technique was the calculation of the reference spectra for ordered hel ix, unordered helix, and sheet, turns, and unordered structures from a seri es of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated s amples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with p revious experimental and theoretical results in the literature. We have dem onstrated the use of these reference spectra for the estimation of secondar y structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinati ons from X-ray crystallography is good. (C) 1999 Academic Press.