Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy.
To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.
Matsuo
Koichi
K
Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima 739-0046, Japan.
Sakurada
Yoshie
Y
Yonehara
Ryuta
R
Kataoka
Mikio
M
Gekko
Kunihiko
K
D000818:Animals; D002942:Circular Dichroism; D004926:Escherichia coli; D006736:Horses; D008786:Metmyoglobin / Q000737:chemistry; D008836:Micrococcal Nuclease / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry
D011506:Proteins; D008786:Metmyoglobin; D013879:Thioredoxins; D008836:Micrococcal Nuclease