Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling.
Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.
Teilum
Kaare
K
Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, Copenhagen, Denmark.
Kragelund
Birthe B
BB
Poulsen
Flemming M
FM
D002942:Circular Dichroism; D026261:Diazepam Binding Inhibitor / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D019791:Guanidine / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D026261:Diazepam Binding Inhibitor; D011994:Recombinant Proteins; D013113:Spin Labels; D019791:Guanidine