Structural and kinetic characterization of early folding events in beta-lactoglobulin.
We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.
Kuwata
K
K
Department of Physiology, School of Medicine, Gifu University, Tsukasa-machi, Gifu 500-8705, Japan.
Shastry
R
R
Cheng
H
H
Hoshino
M
M
Batt
C A
CA
Goto
Y
Y
Roder
H
H
D000818:Animals; D002417:Cattle; D006859:Hydrogen / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D007782:Lactoglobulins; D011522:Protons; D012997:Solvents; D006859:Hydrogen