Structure and function of the conserved domain in alphaA-crystallin. Site-directed spin labeling identifies a beta-strand located near a subunit interface.
Twelve sequential single cysteine mutants of alphaA-crystallin extending between amino acids Y109 and L120 were prepared and reacted with a sulfhydryl specific spin label in order to investigate the role of this sequence in the assembly of the alphaA-crystallin quaternary structure and its chaperone-like function. The sequence is located in the region 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 mobility of the attached nitroxides reveals that the sequence, as a whole, is relatively sequestered from the aqueous solvent. Furthermore, as teh nitroxide is scanned across the sequence, both mobility and accessibility vary with a periodicity of 2, demonstrating that the backbone conformation is that of a beta-strand. Once face of the strand, containing the highly conserved residues R112 and R116, is buried with virtually no accessibility to the aqueous solvent. Equivalent strands from different subunits are in close spatial proximity, as inferred from spin-spin interactions between identical residues along the strand. Taken together, our results are consistent with the hypothesis that the alpha-crystallin domain is a building block of the alpha-crystallins quaternary 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 beta-strand in an unknown structure and demonstrates the feasibility of using this technique to investigate the oligomeric structure of the alpha-crystallins and sHSPs.
Berengian
A R
AR
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Bova
M P
MP
Mchaourab
H S
HS
D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013113:Spin Labels; D013329:Structure-Activity Relationship
D003459:Crystallins; D013113:Spin Labels