Identification of Protein Folding Patterns Using Site-Directed Spin Labeling. Structural Characterization of a β-Sheet and Putative Substrate Binding Regions in the Conserved Domain of αA-Crystallin

• Published in: Biochemistry (Easton) 1998-09, Vol.37 (37), p.12681-12688
• Main Authors: Koteiche, Hanane A, Berengian, Anderee R, Mchaourab, Hassane S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Binding Sites; Circular Dichroism; Conserved Sequence; Crystallins - chemistry; Crystallins - genetics; Cysteine - genetics; Electron Spin Resonance Spectroscopy; Molecular Chaperones - chemistry; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - genetics; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Solvents; Spin Labels
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9814078

The folding pattern of the segment of αA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography were obtained from analysis of the nitroxide mobility and its solvent accessibility. Periodic patterns in the sequence-specific variation of these parameters were used to assign the secondary structure along the sequence. Geometric constraints describing the packing of secondary structure were deduced from patterns of proximities in 20 nitroxide pairs, specifically designed to differentiate between supersecondary structural motifs. Our data, in conjunction with those of Berengian et al. [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951−9957], reveal that the fold of the segment between residues 84 and 120 consists of an antiparallel β-sheet of three strands arranged in consecutive β-hairpins. The boundaries of the sheet are defined at one end by a surface of isologous association and on the other end by an unstructured, charged interdomain segment. One of the putative substrate binding segments overlaps a buried loop, suggesting that the structural origin of the thermal activation of binding is the transient exposure of this site. This paper describes and implements a general strategy for experimental fold recognition using SDSL. The results of its application to αA-crystallin provide the first experimental insight into the folding pattern of the subunit and establish the structural context necessary to understand molecular recognition and substrate binding.