Test of circular dichroism (CD) methods for crambin and CD-assisted secondary structure prediction of its homologous toxins (published erratum appears in Proteins 1990; 7 (3), 296-297)

Journal/Book: Proteins 4 (4), 262-73. 1989;

Abstract: Methods that analyze protein circular dichroism (CD) spectra forfractions of secondary structure are evaluated for the plant proteincrambin, which has a known high-resolution crystal structure. Inaddition, a two-step secondary structure prediction scheme is presentedand used for the toxins homologous to crambin, shown by others to havesecondary structures similar to crambin. The test of CD spectralanalysis methods with the protein crambin employed two computer programsand several CD basis sets. Crambin's crystal structure, known to 0.945Aresolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981),allows accurate evaluation of results. Analysis with the protein spectrabasis sets (Provencher, S.W., Glockner, J. Biochemistry 20:33-37, 1981)as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85,1987) agreed most closely with crambin's crystal structure. This methodwas then applied to the CD spectra of the membrane-active toxinshomologous to crambin (alpha 1- and beta-purothionin, phoratoxin A andB, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") wasdeveloped to assign the secondary structure along the protein chain in ahierarchical fashion and applied to the plant toxins. The methodconstrained the secondary structure fractions to those from CD analysisand combined standard statistical methods with amphipathic helixlocation. Both CD-arrived secondary structure percentages and sequenceassignment indicate that the viscotoxins are structurally most similarto crambin. Purothionin's secondary structure was predicted to befundamentally similar to crambin's with a difference at the start of thefirst helix. This assignment agreed with Raman and NMR analyses ofpurothionin and lends validity to the method presented here. Differencesfrom the NMR in the CD secondary structure fraction analysis forphoratoxin suggest interference in the CD from tryptophan residues.Author.

Keyword(s): AMINO-ACID-SEQUENCE

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