Transient Structure Formation in Unfolded Acyl-coenzyme A-binding Protein Observed by Site-directed Spin Labelling

• Published in: Journal of molecular biology Vol. 324; no. 2; pp. 349 - 357
• Main Authors: Teilum, Kaare, Kragelund, Birthe B, Poulsen, Flemming M
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 22-11-2002
• Subjects: Circular Dichroism; Diazepam Binding Inhibitor - chemistry; Electron Spin Resonance Spectroscopy; Guanidine - pharmacology; Magnetic Resonance Spectroscopy; Mutagenesis, Site-Directed; NMR; Nuclear Magnetic Resonance, Biomolecular; paramagnetic relaxation enhancement; Protein Conformation; Protein Denaturation; Protein Folding; Recombinant Proteins - chemistry; Spectrometry, Fluorescence; spin labelling; Spin Labels; unfolded state; GuHCl, guanidinium chloride; HSQC, heteronuclear single quantum coherence; NMR; paramagnetic relaxation enhancement; unfolded state; ACBP, acyl-coenzyme A-binding protein; MTSL, (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate; spin labelling; protein folding; PRE, paramagnetic relaxation enhancement
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(02)01039-2

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 α-helix formation in the C-terminal part of the polypeptide chain, which forms an α-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.