title: A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins.
abstract: FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy) was used to evaluate the secondary structure of proteins. Four well-studied proteins, concanavalin A, hemoglobin, lysozyme, and trypsin, which have different distributions of secondary structures, were used for assignments of the infrared bands and evaluating the accuracy of FT-IR/PAS methods. Secondary structure contents estimated from FT-IR/PAS and other physical methods (e.g., X-ray diffraction, CD, and traditional FT-IR) show good agreement. In addition, the secondary structure can be evaluated with as little as 0.5 micrograms of protein (concanavalin A), suggesting that FT-IR/PAS is a sensitive and useful technique that could be applied to studies of the folding of recombinant and mutant proteins where only small amounts of material are available. Recombinant phosphorylase kinase gamma 1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase gamma 1-300 subunit has two kinase forms: one has a 10-fold higher activity than the other one. Both fractions, however, are the same as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis. Differences in conformation were demonstrated by using the FT-IR/PAS method, which showed that the low-activity form has more beta-sheet structure than the form with high activity. Analysis of these kinase forms by CD confirms the interpretation made by the FT-IR/PAS method.
authors.affiliation: Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
authors.forename: S
authors.initials: S
authors.lastname: Luo
authors.forename: C Y
authors.initials: CY
authors.lastname: Huang
authors.forename: J F
authors.initials: JF
authors.lastname: McClelland
authors.forename: D J
authors.initials: DJ
authors.lastname: Graves
mesh_terms: D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation
chemical_list: D011994:Recombinant Proteins