Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.
We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.
Smith
S V
SV
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
Correia
J J
JJ
Case
S T
ST
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002683:Chironomidae / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol; D005813:Genes, Synthetic; D019476:Insect Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D017421:Sequence Analysis; D047011:Silk; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D004220:Disulfides; D019476:Insect Proteins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D047011:Silk; D013438:Sulfhydryl Compounds; C095508:rCAS protein, recombinant; D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol