A holistic approach for protein secondary structure estimation from infrared spectra in H2O solutions

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique c ombines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent ba ckground subtraction. Previous attempts to generate structural reference sp ectra from a basis set of reference protein spectra have had limited succes s because of inaccuracies arising from sequential background subtractions a nd spectral normalization, arbitrary spectral band truncation, and attempte d resolution of spectroscopically degenerate structure classes. We eliminat ed these inaccuracies by defining a single mathematical function for protei n spectra, permitting all subtractions, normalizations, and amide band deco nvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with t he sequential nature of previous methods, especially with regard to removin g the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unord ered helix, sheet, turns, and unordered structures from a basis set of spec tra of well-characterized proteins. Structural reference spectra were gener ated in the amide I and amide III bands, both of which have been shown to b e sensitive to protein secondary structure content. We accurately account f or overlaps between amide and nonamide regions and allow different structur e types to have different extinction coefficients. The agreement between ou r structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good. (C ) 2000 Academic Press.