Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.     

Erroneous synthesis of ribosomal proteins in amino acid starved E. coli

The effect of amino acid starvation on the accuracy of translation of ribosomal proteins was analyzed in a stringent (relA+)/relaxed (relA) pair of E. coli strains. The degree of misreading was estimated from the amount of cysteine erroneously incorporated into individual proteins during arginine starvation of bacteria. Illegitimate incorporation of cysteine was found to occur to a significant extent in several proteins from both the small and the large subunits of ribosomes, in either type of strain.     

Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis.

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.     

The role of E. coli single-stranded DNA binding protein in DNA metabolism

Single-stranded DNA binding proteins have been known for some time to be crucial in many DNA metabolic reactions in both prokaryotes and eukaryotes. Despite a wealth of studies on these proteins we still do not understand their biochemical mechanism of action. Recent studies of the Escherichia coli single stranded DNA binding protein (SSB) are beginning to provide some insight into how this and similar proteins might function.     

Cell surface proteins of E. coli

Cell surface proteins of $\text{E. coli}$ K12 have been labelled with ¹²⁵I using a lactoperoxidase method. Results suggest that most outer membrane proteins so far characterised appear to have at least part of their polypeptide chain on the cell surface. These include major outer membrane protains I and II¹, the maltose and vitamin B₁₂ binding proteins and proteins involved in iron transport. The labelling of an antibiotic sensitive mutant and its parent were compared but their labelling patterns did not appear to differ in any way which would suggest the cause of the permeability difference between these two strains.     

Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.     

Exploring the binding dynamics of BAR proteins

We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.     

GroESL proteins facilitate binding of externally added inducer by luxR protein-containing E. coli cells

htpR^? (rpoH, σ32 minus) strain of E. coli harbouring the whole lux system of Vibrio fischeri is very dim. We have recently shown that GroESL proteins fully recover the expression of the lux system in this strain. This work has been undertaken to study our assumption that the GroESL proteins stabilize the LuxR protein, thus enhancing the formation of LuxR–Inducer complex. E. coli htpR^? cells harbouring the luxR gene were unable to bind extracellularly added inducer, while late logarithmically growing htpR⁺ strain bound small quantities of the inducer. Reduction in the nutrient content of the growth medium resulted in a large increase in the capability of these cells to bind the inducer. htpR⁺ or htpR^? E. coli strains harbouring both the luxR and the groESL genes bound large quantities of the inducer. The molecular and ecological significance of these results is discussed.     

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Interpretation of organizational role of proteins on E. coli nucleoid via Hi-C integrated model

Several proteins in Escherichia coli work together to maintain the complex organization of its chromosome. However, the individual roles of these so-called nucleoid-associated proteins (NAPs) in chromosome architectures are not well characterized. Here, we quantitatively dissect the organizational roles of Heat Unstable (HU), a ubiquitous protein in E. coli and MatP, an NAP specifically binding to the Ter macrodomain of the chromosome. Toward this end, we employ a polymer physics-based computer model of wild-type chromosome and their HU- and MatP-devoid counterparts by incorporating their respective experimentally derived Hi-C contact matrix, cell dimensions, and replication status of the chromosome commensurate with corresponding growth conditions. Specifically, our model for the HU-devoid chromosome corroborates well with the microscopy observation of compaction of chromosome at short genomic range but diminished long-range interactions, justifying precedent hypothesis of segregation defect upon HU removal. Control simulations point out that the change in cell dimension and chromosome content in the process of HU removal holds the key to the observed differences in chromosome architecture between wild-type and HU-devoid cells. On the other hand, simulation of MatP-devoid chromosome led to locally enhanced contacts between Ter and its flanking macrodomains, consistent with previous recombination assay experiments and MatP’s role in insulation of the Ter macrodomain from the rest of the chromosome. However, the simulation indicated no change in matS sites’ localization. Rather, a set of designed control simulations showed that insulation of Ter is not caused by bridging of distant matS sites, also lending credence to a recent mobility experiment on various loci of the E. coli chromosome. Together, the investigations highlight the ability of an integrative model of the bacterial genome in elucidating the role of NAPs and in reconciling multiple experimental observations.     

Patterns of E. coli leucine tRNA isoacceptors following bacteriophage MS2 infection.

The RNA extracted from MS2 phage particles can accept radioactive leucine and serine in the presence of tRNA activating enzymes. Leucine acceptance is due to the presence of E. coli leucine tRNA that binds very tightly to the virus particle. RPC-5 column chromatography shows that the pattern of virus associated leucyl-tRNA isoacceptors is different from that of normal E. coli leucyl-tRNA. It is also different from the pattern of host leucyl-tRNA isoacceptors found in E. coli lysate following MS2 phage infection. The RPC-5 pattern of the latter tRNA shows several new peaks of leucine tRNA isoacceptors. The possibility that these tRNAs are some modified forms of normal leucine tRNA isoacceptors is suggested.     

蜡样芽孢杆菌亮氨酸脱氢酶基因在大肠杆菌中的表达及重组酶性质

本研究采用PCR技术从蜡样芽孢杆菌Bacillus cereus基因组DNA中克隆出亮氨酸脱氢酶基因,构建重组表达质粒p ET28α(+)-ldh,实现在大肠杆菌中的高效表达,并分析重组亮氨酸脱氢酶的酶学性质。结果表明,从Bacillus cereus成功克隆的亮氨酸脱氢酶编码基因约为1 000 bp,表达的重组亮氨酸脱氢酶相对分子质量约为40 k Da。酶学研究结果表明:该酶的最适反应温度为37℃,其热稳定性好,30℃的半衰期长达330 h;最适反应p H为9.5;在p H 7.0~8.0的缓冲液中保存24 h后仍保持原有酶活力的80%以上;金属离子Fe2+对该酶具有明显的促进作用,而EDTA强烈抑制亮氨酸脱氢酶的活性。动力学分析结果表明该酶对底物NADH催化的Km和Vmax分别为0.635 mmol/L和1.54μmol/(L·min)。亮氨酸脱氢酶基因在大肠杆菌中的成功表达为手性氨基酸的生物合成提供了可能。