Abstract
K99 pili from bovine strain B44 ofEscherichia coli were incubated in a solution containing 1% sodium dodecyl sulfate (SDS), 4M urea, 1% 2-mercaptoethanol (2-ME), and 10% glycerol at room temperature. After 2 weeks, there was a partial conversion of the pilin from its apparent molecular weight of 17, 000 to 21, 000 due to change in conformation as studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After 10–12 weeks, this change in conformation was complete. The presence of 2-ME in the incubation mixture was essential for this change; this suggested the involvement of a disulfide bond. The role of the disulfide bond could also be shown by reduction and carboxamidation of the pilin. Other modifications such as performic acid oxidation, treatment with iodoacetic acid at acid pH, and 2-hydroxy-5-nitrobenzyl bromide showed that only performic acid oxidation and HNBB modification prevented change in conformation. From these data, it was concluded that the disulfide bond and tryptophan residues of the pilin are essential for the change in conformation in SDS.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature Cited
Brinton CC Jr (1975) The structure, function synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram-negative bacteria Trans NY Acad Sci 27:1003–1054
DeGraaf FK, Klemm P, Gastra W (1981) Purification characterization of the partial covalent structure of the adhesive antigen K99 ofEscherichia coli. Infect Immun 33:877–883
Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 83:70–77
Fish WW, Reynolds JA, Tanford C (1970) Gel chromatography of proteins in denaturing solvents. J Biol Chem 245:5166–5168
Grassetti DR, Murray JF Jr (1967) Determination of sulfhydryl groups with 2,2′-or 4,4′-dithiodipyridine. Arch Biochem Biophys 119:41–49
Gundlach G, Moore S, Stein WH (1959) The reaction of iodoacetate with methionine. J Biol Chem 234:1761–1764
Karkhanis YD, Bhogal BB (1986) A single step isolation of K99 pili fromEscherichia coli. Anal Biochem 155:51–55
Karkhanis YD, Carlo DJ, Zeltner J (1975) A simplified procedure to determine tryptophan residues in proteins. Anal Biochem 69:55–60
Korhonen TK, Nurmiaho E, Ranta H, Eden C (1980) New method for isolation of immunologically pure pili fromEscherichia coli. Infect Immun 27:569–575
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
McMichael JC, Ou JT (1979) Structure of common pili fromEscherichia coli. J Bacteriol 138:969–975
Pitt-Rivers R, Imipiombato FSA (1968) The binding of sodium dodecyl sulfate to various proteins. Biochem J 109:825–830
Salit IE, Gotschlich EC (1977) Hemagglutination by purified type IEscherichia coli pili. J Exp Med 146:1169–1181
Weber K, Osborn M (1969) The reliability of molecular weight-determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Karkhanis, Y.D. Involvement of tryptophan in sodium dodecyl sulfate-induced conformation of K99 pilin. Current Microbiology 15, 73–76 (1987). https://doi.org/10.1007/BF01589364
Issue Date:
DOI: https://doi.org/10.1007/BF01589364