STRUCTURE AND FUNCTION OF THE CONSERVED DOMAIN IN ALPHA-A-CRYSTALLIN - SITE-DIRECTED SPIN-LABELING IDENTIFIES A BETA-STRAND LOCATED NEAR A SUBUNIT INTERFACE
Ar. Berengian et al., STRUCTURE AND FUNCTION OF THE CONSERVED DOMAIN IN ALPHA-A-CRYSTALLIN - SITE-DIRECTED SPIN-LABELING IDENTIFIES A BETA-STRAND LOCATED NEAR A SUBUNIT INTERFACE, Biochemistry, 36(33), 1997, pp. 9951-9957
Twelve sequential single cysteine mutants of alpha A-crystallin extend
ing between amino acids Y109 and L120 were prepared and reacted with a
sulfhydryl specific spin label in order to investigate the role of th
is sequence in the assembly of the alpha A-crystallin quaternary struc
ture and its chaperone-like function. The sequence is located in the r
egion of highest homology in the alpha-crystallin domain, a stretch of
100 amino acids conserved among lens alpha-crystallins and small heat
-shock proteins (sHSPs). Analysis of the solvent accessibility and mob
ility of the attached nitroxides reveals that the sequence, as a whole
, is relatively sequestered from the aqueous solvent. Furthermore, as
the nitroxide is scanned across the sequence, both mobility and access
ibility vary with a periodicity of 2, demonstrating that the backbone
conformation is that of a beta-strand. One face of the strand, contain
ing the highly conserved residues R112 and R116, is buried with virtua
lly no accessibility to the aqueous solvent. Equivalent strands from d
ifferent subunits are in close spatial proximity, as inferred from spi
n-spin interactions between identical residues along the strand. Taken
together, our results are consistent with the hypothesis that the alp
ha-crystallin domain is a building block of the alpha-crystallins quat
ernary structure and suggest that the charge conservation observed in
the alpha-crystallins evolution might be important for the assembly of
the oligomer. This work reports the first use of SDSL to identify a b
eta-strand in an unknown structure and demonstrates the feasibility of
using this technique to investigate the oligomeric structure of the a
lpha-crystallins and sHSPs.