Cyanogen Bromide Fragments Of Protein Research Articles

Major proteins of the outer cell envelope membrane ofEscherichia coli K-12: Multiple species of protein I

Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia+ Ib-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic. Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain. A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.

The major proteins of the Escherichia coli outer cell-envelope membrane. Cyanogen bromide fragments of protein I, composition and order.

The cyanogen bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionine-serine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largely due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced. None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60% of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto.

Cell envelope and shape of Escherichia coli K12. Isolation and preliminary characterization of the major ghost-membrane proteins.

The protein from Escherichia coli ghosts can be separated electrophoretically into three major bands (I, II, and IV) and one minor band (III). The proteins corresponding to bands I, II and IV have been isolated. Protein IV is the lipoprotein described previously by Braun et al. and is thus a homogeneous polypeptide chain. Proteins I and II do not contain phosphorus, palmitic acid, or lipopolysaccharide but small amounts of carbohydrate, probably glucosamine (or a derivative).Proteins I and II have been characterized by amino‐acid analyses, tryptic fingerprints, carboxy‐peptidase digestion, and cyanogen bromide cleavage. Based on cysteic acid the minimum chemical molecular weights of both proteins are the same as those found by electrophoreses: about 40000 (±∼ 10%) for I and about 28000 (±∼ 10%) for II. The numbers of tryptic peptides found do not argue against the assumption that each of the two proteins consists of identical polypeptide chains. However, the sum of the apparent molecular weights of the cyanogen bromide fragments of protein I is almost 80000. The amino acids liberated by carboxypeptidase A at near integral values per 40000 g of protein I correspond to 1 mol each of serine, glycine, alanine, isoleucine, tyrosine, phenylalanine, and to 2 mol valine per 40000 g of protein; however, only 0.5 mol leucine is released. Apart from revealing a rather hydrophobic C‐terminal region, the data thus include the possibility that protein I consists of two different but presumably rather similar polypeptide chains. Protein II is a tryptic fragment of a protein II* with a molecular weight near 40000. Protein II* can give rise to two other fragments by the action of other proteases. The apparent molecular weights of the cyanogen bromide fragments of protein II add up to that of protein II. However, although carboxypeptidase A liberates nearly 1 mol each of serine, alanine, valine, and leucine per 28000 g of protein II, it releases only about 0.5 mol each of glutamic acid, glycine and isoleucine. As for protein I, it cannot yet be excluded that protein II consists of two polypeptide chains. If so, the two must be very similar. Regardless as to whether proteins I or II consist of one or two polypeptide chains these must be arranged as repeating subunits in the outer membrane of the E. coli cell envelope.The properties of proteins I and II allow them to be classified as intrinsic or integral membrane proteins.

Cell envelope and shape of Escherichia coli K12. The ghost membrane.

Rod‐shaped “ghosts” can be isolated from Escherichia coli (and a number of other gram‐negative organisms) which are devoid of murein, are surrounded by a unit membrane and have lost all intracellular material except for some internal membrane which is derived from the cytoplasmic membrane. Ghosts consist of about 25% phospholipid, 25–30% lipopolysaccharide and 45–50% protein.Dodecylsulfate‐gel electrophoresis resolves the protein into four main bands with the apparent molecular weights: I, 40000, II, 28000, III, 18000 and IV, 10000. The proteins of all four bands are predominantly located in the outer membrane of the cell wall. Band IV has been identified as the lipoprotein, part of which is bound covalently to murein [1]. Band II very likely represents a homogeneous polypeptide chain and is derived by the action of trypsin on ghosts from a protein II* which has a molecular weight of about 40000. The molar ratio of polypeptides I, II* and IV in ghosts is about 1:1:8–10. Protein of band III is present in smaller amounts which, in contrast to I, II* and IV, were found to vary from preparation to preparation.Proteins I, II* and IV comprise 7–10% of the total cellular protein and they are most likely arranged in the outer membrane of the cell envelope as repeating subunits. Several lines of evidence indicate that proteins of all four bands are also present in the cytoplasmic membrane, although in considerably lower quantities than in the outer membrane. The synthesis of these cell wall proteins does not require DNA or murein synthesis.The phospholipid in ghosts is not required for their macroscopic structural integrity or for the unit membrane appearance of either of their membrane systems. A phospholipid bilayer is therefore not the basic structure of the ghost membranes.