Singular value decomposition with self-modeling applied to determine bacteriorhodopsin intermediate spectra: Analysis of simulated data

An a priori model-independent method for the determination of accurate spec tra of photocycle intermediates is developed, The method, singular value de composition with self-modeling (SVD-SM), is tested on simulated difference spectra designed to mimic the photocycle of the Asp-96 --> Asn mutant of ba cteriorhodopsin. Stoichiometric constraints, valid until the onset of the r ecovery of bleached bacteriorhodopsin at the end of the photocycle, guide t he self-modeling procedure. The difference spectra of the intermediates are determined in eigenvector space by confining the search for their coordina tes to a stoichiometric plane. In the absence of random noise, SVD-SM recov ers the intermediate spectra and their time evolution nearly exactly, The r ecovery of input spectra and kinetics is excellent although somewhat less e xact when realistic random noise is included in the input spectra, The diff erence between recovered and input kinetics is now visually discernible, bu t the same reaction scheme with nearly identical rate constants to those as sumed in the simulation fits the output kinetics well. SVD-SM relegates the selection of a photocycle model to the late stage of the analysis. It thus avoids derivation of erroneous model-specific spectra that result from glo bal model-fitting approaches that assume a model at the outset.