title: Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structures.
abstract: Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.
authors.affiliation: Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York 10029.
authors.forename: S J
authors.initials: SJ
authors.lastname: Prestrelski
authors.forename: D M
authors.initials: DM
authors.lastname: Byler
authors.forename: M N
authors.initials: MN
authors.lastname: Liebman
mesh_terms: D000818:Animals; D002417:Cattle; D005583:Fourier Analysis; D046911:Macromolecular Substances; D011487:Protein Conformation; D012996:Solutions; D013055:Spectrophotometry, Infrared / Q000379:methods; D014357:Trypsin / Q000737:chemistry*; D014362:Trypsinogen / Q000737:chemistry; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D046911:Macromolecular Substances; D012996:Solutions; D014362:Trypsinogen; D014357:Trypsin