Microsecond melting of a folding intermediate in a coiled-coil peptide, monitored by T-jump/UV Raman spectroscopy.
A truncated version of the GCN4 coiled-coil peptide has been studied by ultraviolet resonance Raman (UVRR) spectroscopy with 197 nm excitation, where amide modes are optimally enhanced. Although the CD melting curve could be satisfactorily described with a two-state transition having Tm = 30 degrees C, singular value decomposition of the UVRR data yielded three principal components, whose temperature dependence implicates an intermediate form between the folded and unfolded forms, with formation and melting temperatures of 10 and 40 degrees C. Two alpha-helical amide III bands, at 1340 and 1300 cm(-1), melted out selectively at 10 and 40 degrees C, respectively, and are assigned to hydrated and unhydrated helical regions. The hydrated regions are proposed to be melted in the intermediate form, while the unhydrated regions are intact. Time-resolved UVRR spectra following laser-induced temperature jumps revealed two relaxations, with time constants of 0.2 and 15 mus. These are suggested to reflect the melting times of hydrated and unhydrated helices. The unhydrated helical region may be associated with a 14-residue "trigger" sequence that has been identified in the C-terminal half of GCN4. Dehydration of helices may be a key step in the folding of coiled-coils.
Balakrishnan
Gurusamy
G
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Hu
Ying
Y
Case
Martin A
MA
Spiro
Thomas G
TG
D000465:Algorithms; D020816:Amino Acid Motifs; D050976:Basic-Leucine Zipper Transcription Factors; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D044367:Phase Transition; D025341:Principal Component Analysis; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry*; D044366:Transition Temperature; D014867:Water / Q000737:chemistry
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014867:Water