Factors affecting the efficiency of protein synthesis in Escherichia coli. Production of a polypeptide of more than 6000 amino acid residues

Factors affecting the efficiency of protein synthesis were analyzed in Escherichia coli. For this purpose the lacZ gene was fused to produce polypeptides from a dimer (molecular weight 229,957) to a hexamer (molecular weight 684,924) of beta-galactosidase. From pulse-chase experiments it was found that only 45% of the ribosomes which reached to the end of the first monomer were able to complete the second monomer unit. Similarly, for every addition of a monomer unit to synthesize the multimers from the trimer to the hexamer approximately half of the ribosomes failed to complete the synthesis of the added unit. Furthermore, the stability of the polypeptides decreased as their sizes increased. As a result, the overall efficiency of the production of the beta-galactosidase polymers dropped by a factor of approximately 3 on a weight basis for each addition of a monomer unit.     

Differential scanning calorimetry of lobster haemocyanin

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.     

Phycocyanin aggregation. A small angle neutron scattering and size exclusion chromatographic study

The influence of environmental factors on the aggregation properties of phycocyanin from Synechocystis 6701 was studied by small angle neutron scattering and high-pressure size-exclusion liquid chromatography. Phycocyanin was found to exist in a reversible equilibrium between the monomer, trimer and hexamer forms. The distribution of the protein between these oligomers is determined by the pH, buffer composition and ionic strength of the medium, and protein concentration. Phycocyanin was in a stable hexameric state at pH 5.0 to 6.0 at a concentration of 1 to 10 mg/ml, and was primarily in a trimeric state at pH 8.0 at a concentration of about 5 mg/ml. Comparison of the small angle scattering data with the computed scattering curve for a hollow cylinder was used to determine the dimensions of the best-fit model by a least-squares fitting procedure. The outer radius, inner radius and height of the phycocyanin hexamer were found to be 54.1, 12.0 and 61.4 A (1 A = 0.1 nm), respectively, and the corresponding dimensions for the trimer were 54.5, 14.0 and 33.0 A. The molecular weight ratio for phycocyanin hexamer was determined to be 217,000. The dimensions and molecular weight ratios of phycocyanin from Synechocystis 6701 obtained by solution scattering are similar to the values for Mastigocladus laminosus obtained by X-ray crystallography.     

The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.     

Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase

The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.     

Molecular weight and shape of the phycocyanin hexamer

The hexamer of phycocyanin from Phormidium luridum has been isolated and purified by ammonium sulfate fractionation and gel chromatography. The protein is characterized by the sedimentation constant S°_{20, w} = 10.2S, the diffusion coefficient D_{20, w} = 4.73 × 10^?7 cm²/sec, and intrinsic viscosity [η] = 3.89 ml/g. The molecular weight of the aggregate is 209,000. The shape and dimensions of the hexamer are discussed in terms of a model consisting of subunits arranged with C₆ symmetry. The monomers, assumed to be spherical, are found to have a radius of 22 Å, and the diameter across the hexamer is 132 Å. The latter figure agrees closely with dimensions observed in electron micrographs.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Morpheeins--a new structural paradigm for allosteric regulation

Classic models for the allosteric regulation of protein function consider an equilibrium among protein structures of constant oligomeric multiplicity. The morpheein (mor-phee'-in) concept expands this model to include a dynamic equilibrium of protein structures wherein a protein monomer can exist in more than one conformation and each monomer conformation dictates a different quaternary structure of finite multiplicity and different functionality. The morpheein concept provides a new framework for understanding allosteric regulation, kinetic cooperativity and hysteresis. Porphobilinogen synthase constitutes a prototype morpheein ensemble comprising several interconverting quaternary structure isoforms; one monomer conformation dictates assembly of a high-activity octamer, whereas an alternative monomer conformation dictates assembly of a low-activity hexamer. It is proposed here that the behavior of some other allosteric enzymes reflect dynamic morpheein equilibrium systems and six candidate proteins are enumerated.     

Anisotropy of the fluorescence from the monomer,five oligomers,and a polymer of epicatechin

The steady-state fluorescence anisotropy r has been measured at 25°C for dilute solutions of epicatechin units connected by interflavan bonds with 4β → 8 stereochemistry. The molecules studied are monodisperse oligomers from the monomer through the hexamer, and a polydisperse sample with a number-average degree of polymerization x_n of 10. The hexamer and smaller oligomers have been removed from the polydisperse sample. All samples have a very small value of r in 1,4-dioxane and in methanol. Higher values of r are seen in ethylene glycol and in glycerol. In the latter two solvents, the monomer has significantly higher r than any species with x_n > 1. This observation is interpreted as evidence for the presence of excitation migration from one monomer to another in the dimer and species of higher x_n.     

胰岛素六聚体在水溶液中的构象柔性研究

用分子动力学（MD)模拟方法设计了两个模拟时间为600ps的对比计算机模拟实验,研究了R6态的胰岛素六聚体在水溶液中的构象柔性。通过对MD模拟所得到的轨迹的分析发现,包含锌离子和苯酚的胰岛素六聚体体系的构象柔性弱于不含锌离子和苯酚的胰岛素六聚体体系,对于不包含锌离子和苯酚的体系,胰岛素六聚体的构象柔性表现得较为突出,特别是在实验研究认为与胰岛素和受体结合位点有关的每个单体的B链羧端的β折叠部分,发生了快速而显著的构象变化,表现出了很大的构象柔性。这些模拟结果与实验观测结果相吻合。     

Complete amino acid sequence of jack bean urease

The subunit structure of jack bean urease has been unresolved in spite of many investigations. Thus far, the molecular weight for the native urease seem to range from 480,000 to 590,000 and the values for the monomer range from 30,000 to 97,000. The complete amino acid sequence of jack bean urease has been determined primarily by sequencing cyanogen bromide peptides, which were aligned by overlapping peptides obtained by lysylendopeptidase digestion of the protein and tryptic digestion of the citraconylated protein. The protein contains 840 amino acid residues in a single polypeptide chain and the subunit molecular weight calculated from the sequence is 90,790. The value of 544,740 for the hexamer, consistent with the value of 580,000 determined for intact urease by centrifugal analyses, indicated that urease consists of six subunits. Thirteen of 25 histidine residues in the urease subunit are crowded in the region between residues 479 and 607. Urease is a nickel metalloenzyme and the nickel has an essential role in catalysis by this enzyme. It is noteworthy that cysteine-592, which is recognized as essential for enzymatic activity and is related to the nickel ion in the active center, is located on this histidine-rich sequence.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Solubilization of polyaromatic hydrocarbons by recombinant bioemulsifier AlnA

AlnA is the protein responsible for the emulsifying and solubilizing activity of the Acinetobacter radioresistens KA53 bioemulsifier alasan. AlnA was produced in Escherichia coli, purified to homogeneity and then used to measure the enhanced solubility of 12 polyaromatic hydrocarbons (PAHs). The amount of PAH solubilized was directly proportional to AlnA concentration. The ratio of PAH solubilized by 40 μg/ml AlnA compared to that soluble in the aqueous buffer varied greatly, from 4 (fluorene) to 81 (hexylbenzylcyclosilane). Calculations of moles PAH solubilized per mole AlnA yielded values from 4.3 (hexylphenylbenzene) to 55.8 (1,10-phenanthrolene). There was no obvious relationship between the amount of PAH solubilized and its molecular weight or intrinsic solubility. Native gel electrophoresis indicated that AlnA formed hexamers in the presence of PAHs. With molar ratios of fluorene to AlnA of 0.75 or less, only the monomer was observed, whereas at ratios of 7.5 or higher, only the hexamer was detected. At an intermediate molar ratio of 2.6, both monomer and hexamer appeared. The data indicate that PAHs are initially solubilized by binding to the monomeric form of AlnA, and as the amount bound increases above one molecule PAH per AlnA, the protein aggregates to form a specific oligomer of 5–8 monomers which allows for the binding and solubilization of more PAH. Electronic Publication     

Secondary structure and assembly mechanism of an oligomeric channel protein

The alpha-toxin of Staphylococcus aureus is secreted as a water-soluble, monomeric polypeptide (Mr 33 182) that can assemble into an oligomeric membrane channel. By chemical cross-linking, we have confirmed that the major form of the channel is a hexamer. The circular dichroism spectrum of this hexamer in detergent revealed that it contains a high proportion of beta-sheet that we deduce must lie within the lipid bilayer when the protein is associated with membranes. The circular dichroism spectrum of the monomeric toxin in the presence or absence of detergent was closely similar to the spectrum of the hexamer, suggesting that the secondary structure of the polypeptide is little changed on assembly. Results of experiments involving limited proteolysis of the monomer and hexamer are consistent with the idea that assembly involves the movement of two rigid domains about a hinge located near the midpoint of the polypeptide chain. The hydrophilic monomer is thereby converted to an amphipathic rod that becomes a subunit of the hexamer.     

Examination of the dynamic assembly equilibrium for E. coli ClpB

Escherichia coli ClpB is a heat shock protein that belongs to the AAA+ protein superfamily. Studies have shown that ClpB and its homologue in yeast, Hsp104, can disrupt protein aggregates in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity. In addition, it is widely assumed that ClpB is uniformly hexameric in the presence of nucleotides. Here we report, in the absence of nucleotide, that increasing ClpB concentration leads to ClpB hexamer formation, decreasing NaCl concentration stabilizes ClpB hexamers, and the ClpB assembly reaction is best described by a monomer, dimer, tetramer, hexamer equilibrium under the three salt concentrations examined. Further, we found that ClpB oligomers exhibit relatively fast dissociation on the time scale of sedimentation. We anticipate our studies on ClpB assembly to be a starting point to understand how ClpB assembly is linked to the binding and disaggregation of denatured proteins. Proteins 2015; 83:2008–2024. © 2015 Wiley Periodicals, Inc.     

Synchrotron protein footprinting supports substrate translocation by ClpA via ATP-induced movements of the D2 loop

Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.     

Novel hexamerization motif is discovered in a conserved cytoplasmic protein from <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 A. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long beta-hairpin protrusion that distinguishes this structure. This beta-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.