Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid

Abstract

The primary structure of a covalent lipoglycoprotein (murein . lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of57 residues. The lipid is attached to the N-terminal serine and the murein is bound to the 6-amino group of the C-terminal lysine of the lipoprotein. The repetitive design of the sequence suggcsts a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occurred.This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2O/,, 0.5O/, and O.lo/o dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of I0000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bound to the lipoprotein. This was not due to a different amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod-like shape of standard proteins in dodecylsulfate.The lipoprotein which is covalently attached to the murein [I] of the outer membrane of Escherichia coli [2--51 is one of thc few membrane proteins isolated so far in pure form. Even soluble lipoproteins, as they occur e.g. in the serum, are difficult to purify and only rather limited information about their structure is available today [6,7]. In addition, in the lipoprotein we are working, the lipid is very probably covalently bound [2,8] which was not found in any other lipoprotein mixture studied so far. I n order to elucidate the attachment site of the lipid and the mode of binding on the murein, we studied the amino acid sequence of the polypeptide chain. In a previous paper [Y] we discussed some features of the unusual and very interesting results especially with regard to the repetitive design of the sequence and the relevance of this study to the structure of membrane proteins and membranes in general. Now we present the experimental evidence together with a full account of the results.

Characterization of the free form of murein‐lipoprotein from the outer membrane of Escherichia coli B/r

“…The amino acid composition of the free lipoprotein was found to be the same as the amino acid composition calculated from the amino acid sequence of the murein-bound lipoprotein [8,9] except for the presence of 0.25 mol diaminopimelate and an excess of about 0.5 mol glutamic acid per mole protein. In confirmation of the data obtained by Hirashima et al [3], there were no traces of muramic acid or glucosamine, and the diaminopimelate and the excess glutamate may be due to the action of an unknown protease during isolation.…”

Mutants of Escherichia coli K12 lacking all ‘major’ proteins of the outer cell envelope membrane

“…The commonly used designation 'major' proteins for the proteins discussed here is somewhat misleading in that the 10 000 daltons lipoprotein studied in detail by Braun and collaborators [21] clearly also constitutes a major outer membrane protein. The mutants described here do not lack the lipoprotein.…”

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

“…They studied Lpp in E. coli. It had been known for some time that Lpp existed in two distinct forms -one covalently bound to the peptidoglycan, and a 'free' population that did not interact with peptidoglycan (Braun & Rehn, 1969;Braun & Bosch, 1972). The novel observation made by Cowles et al (2011) was the discovery that the 'free' population of Lpp spans the outer membrane, with one end of its trimeric structure exposed to the extracellular space.…”