TY - JOUR
T1 - A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins
AU - Luo, Siquan
AU - Huang, Chi Ying F.
AU - McClelland, John F.
AU - Graves, Donald J.
PY - 1994/1
Y1 - 1994/1
N2 - 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 μg 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 γ1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase γ1-300 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 β-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.
AB - 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 μg 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 γ1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase γ1-300 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 β-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.
UR - http://www.scopus.com/inward/record.url?scp=0028013209&partnerID=8YFLogxK
U2 - 10.1006/abio.1994.1009
DO - 10.1006/abio.1994.1009
M3 - Article
AN - SCOPUS:0028013209
SN - 0003-2697
VL - 216
SP - 67
EP - 76
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -