Invariant tryptophan at a shielded site promotes folding of the conformational unit of spectrin.
The tryptophan that is highly conserved among repeating structural units of spectrin is reported to promote the conformational stability of one such unit of chicken brain alpha-spectrin. Four constructs were inserted into pET vectors for overexpression in Escherichia coli of the following spectrin peptides: (i) two adjacent but separately expressed "conformationally phased" repeating units, R16 and R17, one of which (R17) contains a single tryptophan; (ii) a mutant, M17, of the single tryptophan-containing unit with alanine substituted for the tryptophan; and (iii) a conformationally unphased unit, 1617, composed of half of each of the phased units. Both the mutant unit and the unphased unit were much more readily digested by chymotrypsin and by elastase than the phased units and exhibited only 38% and 54% as much alpha-helical structure, respectively, as the phased units by their far UV CD spectra; 90 degrees light scattering measurements revealed the folded peptides to be predominantly monomeric in solution, whereas the unfolded, protease-sensitive peptides consisted of dimers and/or trimers. This trend was corroborated by their dynamic light scattering. Both the blue-shifted wavelength of maximal emission and the relative inaccessibility to acrylamide of the single tryptophan in the folded unit indicate that the invariant tryptophan occupies a site that is shielded from the aqueous phase.
MacDonald
R I
RI
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Musacchio
A
A
Holmgren
R A
RA
Saraste
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013049:Spectrin; D014364:Tryptophan; D002918:Chymotrypsin; D010196:Pancreatic Elastase