IDENTIFICATION OF PROTEIN-FOLDING PATTERNS USING SITE-DIRECTED SPIN-LABELING - STRUCTURAL CHARACTERIZATION OF A BETA-SHEET AND PUTATIVE SUBSTRATE-BINDING REGIONS IN THE CONSERVED DOMAIN OF ALPHA-A-CRYSTALLIN
Ha. Koteiche et al., IDENTIFICATION OF PROTEIN-FOLDING PATTERNS USING SITE-DIRECTED SPIN-LABELING - STRUCTURAL CHARACTERIZATION OF A BETA-SHEET AND PUTATIVE SUBSTRATE-BINDING REGIONS IN THE CONSERVED DOMAIN OF ALPHA-A-CRYSTALLIN, Biochemistry (Easton), 37(37), 1998, pp. 12681-12688
The folding pattern of the segment of alpha A-crystallin encoded by ex
on 2 and containing putative substrate binding sites was explored usin
g site-directed spin labeling (SDSL). For this purpose, a nitroxide sc
an was carried out between residues 60 and 108. At each site, structur
al constraints describing the local environment and topography were ob
tained from analysis of the nitroxide mobility and its solvent accessi
bility. Periodic patterns in the sequence-specific variation of these
parameters were used to assign the secondary structure along the seque
nce. Geometric constraints describing the packing of secondary structu
re were deduced from patterns of proximities in 20 nitroxide pairs, sp
ecifically designed to differentiate between supersecondary structural
motifs, Our data, in conjunction with those of Berengian et al. [Bere
ngian, 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 that the fold or the segment between residues 84 a
nd 120 consists of an antiparallel beta-sheet of three strands arrange
d in consecutive beta-hairpins. The boundaries of the sheet are define
d at one end by a surface of isologous association and on the other en
d by an unstructured, charged interdomain segment. One of the putative
substrate binding segments overlaps a buried loop, suggesting that th
e structural origin of the thermal activation of binding is the transi
ent exposure of this site. This paper describes and implements a gener
al strategy for experimental fold recognition using SDSL. The results
of its application to alpha A-crystallin provide the first experimenta
l insight into the folding pattern of the subunit and establish the st
ructural context necessary to understand molecular recognition and sub
strate binding.