Conformational Studies on Murein‐Lipoprotein from the Outer Membrane of Escherichia coli

Abstract

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of the Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein IT) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an a-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64 % a-helix and 15 % p-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5-12. Denaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 "C for lipoprotein I and at 85.1 "C for lipoprotein 11. Lipoprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein conformation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.The conformation of membrane proteins has played an important role in discussions on the molecular organization of biological membranes [I -41. The outer membrane of Escherichia coli is basically constructed like other biological membranes in that glycolipids are at the surface, and phospholipids and proteins are the major constituents. A peculiarity of the bacterial cell's outer layer is the murein net composed of polysaccharide chains cross-linked by short peptide side-chains [5]. Lipoprotein molecules of a single type are covalently fixed at the murein net and extend into the outer membrane [6,7]. Together with the free form of lipoprotein not covalently bound to the murein [8], they constitute the most abundant protein in E. coli. It can be assumed that certain properties of the outer membrane, such as its function as a permeability barrier against certain substrates and antibiotics, are determined substantially by this lipoprotein. Knowledge of the lipoprotein's conformation would aid in the construction of a tentative model of outer membrane structure.Immunological studies with the aim of localizing the lipoprotein within the outer membrane showed that lipoprotein acts as an antigen in mutant cells with defects in cell surface structures and that in wild-type cells it is buried in the membrane (9 -111. Since the amino acid sequence of the lipoprotein is known [12,13], we could study the regions of the polypeptide chain against which antibodies were directed. From the two lipoprotein samples used for the conformational studies in this paper, one was released from the murein by degradation of murein with lysozyme and the other by cleaving the polypeptid...

Antigenic Determinants of Murein Lipoprotein and Its Exposure at the Surface of Enterobacteriaceae

“…Even treatment with a boiling solution of 4 % sodium dodecylsulfatc and subsequent precipitation by acetone, both agents known to denature proteins, are without major effect on the antigenic specificity of the lipoprotein. From a detailed study on the conformation of lipoprotein [13] we know that it easily renatures in buffer solution even after extensive denaturation.…”

“…The contribution of the muropeptide units to the antigenic sites of lipoprotein I was tested by releasing the muropeptides from the lipoproein by trypsin treatment [13]. The preferential cleavage sites of trypsin are located at the C-terminal end of lipoprotein as indicated by the arrows in Fig.…”

“…The preparation of the various lipoprotein samples has been described in detail elsewhere : lipoprotein I (containing two or three muropeptides) is obtained by degradation of murein with lysozyme [12]; lipoprotein I1 (i.e. free of muropeptides and lacking the C-terminal peptide Tyr-Arg-Lys) is released from murein by treatment with trypsin [13]; isolation of lipoprotein IV (i.e. free lipoprotein occurring in the outer membrane, not bound to murein) is obtained according to Hindenach and Henning 1141.…”

“…The protein content of samples was either determined after acid hydrolysis using the amino acid analyzer as described previously [13] or after alkaline hydrolysis (2.5 M NaOH for 2.5 h at 90 "C) with ninhydrin [18]. The samples were adjusted to pH 5.5 by adding 1 ml 30% acetic acid.…”

Activation of Lymphocyte Populations with Concanavalin A or with Lipoprotein and Lipopeptide from the Outer Cell Wall of Escherichia coli Correlation of Early Membrane Changes with Induction of Macromolecular Synthesis

“…In short, cells were disrupted with glass beads and the cell envelope was separated by differential centrifugation and solubilized in 4 % boiling sodium dodecyl sulphate. The murein-lipoprotein complex was spun down and the mureinfree form of lipoprotein was obtained from the supernatant according to the method of Braun et al [9]. The lipopolysaccharide content of the preparations was below 1 % as judged from their content of /3-hydroxymyristic acid, which occurs in lipopolysaccharide but not in lipoprotein [5].…”