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Results for “Protein Multimerization”

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PubMed1992

A functional protein hybrid between the glucose transporter and the N-acetylglucosamine transporter of Escherichia coli.

The glucose and N-acetylglucosamine-specific transporters (IIGlc/IIIGlc and IIGlcNAc) of the bacterial phosphotransferase system mediate carbohydrate uptake across the cytoplasmic membrane concomitant with substrate phosphorylation. The two transporters have 40% amino acid sequence identity. Eight chimeric proteins between the two transporters were made by gene reconstruction. All hybrid proteins could be expressed, some inhibited cell growth, and one was active. The active hybrid transporter consists of the transmembrane domain (residues 1-386) of the IIGlc subunit and the two hydrophilic domains (residues 370-648) of IIGlcNAc. The N-terminal hydrophilic domain of IIGlcNAc contains the transiently phosphorylated cysteine-412. The hybrid protein is specific for glucose, which indicates that the sugar specificity determinant is in the transmembrane domain and that the cysteine from which the phosphoryl group is transferred to the substrate is not part of the binding site. The protein sequence (LKTPGRED) at which the successful fusion occurred has the characteristic properties of an interdomain oligopeptide linker (Argos, P., 1990, J. Mol. Biol. 211, 943-958).

Acetylglucosamine↗

PubMed1988

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli.

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.

ATP-Binding Cassette Transporters↗

PubMed1987

Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells.

We report here for the first time reconstitution and secretion of functionally active antibody in non-lymphoid cells. Expression vectors for the light and the heavy chain of a monoclonal antibody directed against creatine kinase (EC 2.7.3.2) were introduced into COS and CHO Chinese hamster ovary dhfr- cells. Introduction of the expression vectors separately gave rise to immuno-reactive material in the culture supernatants, but only cotransfection of the expression plasmids resulted in secretion of protein with immuno-reactivity against antibodies directed against mouse heavy and light chains as well as specific antigen-binding affinity, as determined by enzyme-linked immunosorbent assay. Secreted kappa and gamma chains from reconstituted antibody were characterized by immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In COS cells, reconstituted antibody was transiently secreted; cotransfection of kappa and gamma chain expression plasmids with a dihydrofolate reductase (DHFR)-expression plasmid into CHO dhfr- cells gave rise to stable transformants secreting functionally active antibody.

Animals↗

PubMed2024

Rabies Virus Phosphoprotein Exhibits Thermoresponsive Phase Separation with a Lower Critical Solution Temperature.

Rabies virus (RABV) generates membrane-less liquid organelles (Negri bodies) in the cytoplasm of its host cell, where genome transcription and replication and nucleocapsid assembly take place, but the mechanisms of their assembly and maturation remain to be explained. An essential component of the viral RNA synthesizing machine, the phosphoprotein (P), acts as a scaffold protein for the assembly of these condensates. This intrinsically disordered protein forms star-shaped dimers with N-terminal negatively charged flexible arms and C-terminal globular domains exhibiting a large dipole moment. Our study shows that in vitro self-association of RABV P drives a complex thermoresponsive phase separation with a lower critical solution temperature. Protein dimers assemble already below the saturation concentration, and condensation is driven by attractive conformation-specific interactions leading to reentrant liquid phase separation over a narrow range of salt concentration. We propose a minimal molecular model in which P can adopt three limit conformational states and the disordered N-terminal arms control the interactions between giant dipoles that is consistent with our observations.

Rabies virus↗

PubMed2025

The C-terminal Domain of SARS-CoV-2 nsp8 is a Molten Globule in the Absence of Binding Partners.

The coronavirus genome is transcribed by a replication-transcription complex (RTC) containing the RNA polymerase plus additional cofactors. The cofactor nsp8 is an important component of the RTC in both alpha and betacoronaviruses required for nsp12 polymerase activity, complex stability, and recruitment of other RTC cofactors. Here we use NMR and other biophysical methods to characterize the structural features and oligomeric state of full-length nsp8 in solution. We show that the C-terminal domain of nsp8 has molten-globule like intrinsic disorder, while the N-terminal domain retains its folded structure in the absence of binding partners. Our data also shows a concentration-dependent association of nsp8 into dimers and possibly tetramers, but not larger molecular weight species. Upon binding nsp7, the C-terminal domain of nsp8 folds into a well-defined conformation consistent with available structures of the complex, while the linker region connecting the N- and C-terminal domains remains disordered.

Viral Nonstructural Proteins↗

PubMed1989

Production in eukaryotic cells and characterization of four hybrids of tissue-type and urokinase-type plasminogen activators.

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.

Animals↗

PubMed1991

A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120.

Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development.

Amino Acid Sequence↗

PubMed1992

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.

Amino Acid Sequence↗

PubMed1987

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

The location of exon boundaries in the multimeric iron-storage protein ferritin.

The nature of the amino acids whose codons border introns in ferritin genes is novel; the disposition of these intron boundaries within the three-dimensional structure of the 24-subunit molecule differs significantly from that of other proteins. These observations are discussed in relation to the functions of isoferritins.

Amino Acid Sequence↗

PubMed1986

Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins.

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.

Ferrous Compounds↗

PubMed1986

Properties of chemically modified Ni(II)-Fe(II) hybrid hemoglobins. Ni(II) protoporphyrin IX as a model for a permanent deoxy-heme.

Chemical modifications, NES-Cys(beta 93), des-Arg(alpha 141), and both modifications on the same molecule, were made to Ni-Fe hybrid hemoglobins, and their effect on individual subunits was investigated by measuring oxygen equilibrium curves, the Fe(II)-N epsilon (His F8) stretching Raman lines, and light-absorption spectra. The oxygen equilibrium properties indicated that modified Ni-Fe hybrid hemoglobins remain good models for the corresponding deoxy ferrous hemoglobins, although K1, the dissociation equilibrium constant for the first oxygen to bind to hemoglobin, was decreased by the chemical modifications. Resonance Raman spectra of deoxy alpha 2 (Fe) beta 2 (Ni) and light-absorption spectra of deoxy alpha 2 (Ni) beta 2 (Fe), revealed that the state of alpha hemes in both hybrid hemoglobins underwent a transition from a deoxy-like state to an oxy-like state caused by these chemical modifications when K1 was about 3 mm Hg (1 mm Hg approximately 133.3 Pa). On the other hand, the state of beta hemes in hybrid hemoglobins was little affected, when K1 was larger than 1 mm Hg. Modified alpha 2 (Fe) beta 2 (Ni) gave a Hill coefficient greater than unity with a maximum of 1.4 when K1 was about 4 mm Hg. The two-state model predicts that the K1 value at the maximum Hill coefficient should be much larger than this value. For oxygen binding to unmodified alpha 2 (Ni) beta 2 (Fe), oxygen equilibrium data suggested no structural change, while the spectral data showed a structural change around Ni(II) protoporphyrin IX in the alpha subunits. A similar situation was encountered with modified alpha 2 (Ni) beta 2 (Fe), although K1 was decreased as a result of the structural changes induced by the modifications.

Ferrous Compounds↗

PubMed1990

Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.

Amino Acid Sequence↗

PubMed1990

Effects of expression of mammalian G alpha and hybrid mammalian-yeast G alpha proteins on the yeast pheromone response signal transduction pathway.

Scg1, the product of the Saccharomyces cerevisiae SCG1 (also called GPA1) gene, is homologous to the alpha subunits of G proteins involved in signal transduction in mammalian cells. Scg1 negatively controls the pheromone response pathway in haploid cells. Either pheromonal activation or an scg1 null mutation relieves the negative control and leads to an arrest of cell growth in the G1 phase of the cell cycle. Expression of rat G alpha s was previously shown to complement the growth defect of scg1 null mutants while not allowing mating. We have extended this analysis to examine the effects of the short form of G alpha s (which lacks 15 amino acids present in the long form), G alpha i2, G alpha o, and Scg1-mammalian G alpha hybrids. In addition, we have found that constructs able to complement scg1 are also able to inhibit the response to pheromone and mating when expressed in a wild-type SCG1 strain. Overexpression of Scg1 has a similar inhibitory effect. These results are consistent with a model proposed for the action of Scg1 as the alpha component of a heterotrimeric G protein in which the beta gamma component (Ste4/Ste18) activates the pheromone response after dissociation from Scg1. They suggest that the G alpha constructs able to complement scg1 can interact with beta gamma to prevent activation of the pathway but are unable to interact with pheromone receptors to activate the pathway.

Animals↗

PubMedDate not supplied

Selective killing of laryngeal carcinoma cells by a monoclonal immunotoxin.

To overcome the lack of selectivity of present anticancer drugs, an alternative approach is described for laryngeal carcinoma cells. An immunotoxin is synthesized containing the toxic chain from ricin coupled to a monoclonal antibody against a laryngeal carcinoma cell line. The newly formed monoclonal immunotoxin selectively kills cells from laryngeal carcinoma cell lines, whereas various normal living cells are not affected. Control experiments rule out the possibility that toxicity can be attributed to contaminating traces of whole ricin toxin. The results indicate that minute amounts of the immunotoxin are selectively cytotoxic for living laryngeal carcinoma cells in vitro.

Antibodies, Monoclonal↗

PubMed1992

Functional dissection of AP-1 transcription factors using the Gal4 adaptor assay.

In the Gal4 adaptor assay, leucine zipper-containing proteins are tethered indirectly to the promoters of Gal4-responsive reporter genes via synthetic protein chimeras consisting of a Gal4 DNA-binding domain with an attached leucine zipper. Ternary complexes composed of the DNA binding site, the adaptor protein, and a leucine zipper factor can stimulate reporter gene activity, provided that the latter component possesses a transcriptional activation domain. This system is used to assay the transcriptional function and the interactions between various AP-1 factors.

Amino Acid Sequence↗

PubMed1992

Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits.

We have used the mouse alpha (alpha M) and human alpha (alpha H) subunits to investigate the molecular mechanisms of assembly of the mammalian acetylcholine receptor (AChR) transiently expressed in COS cells. COS cells expressing hybrid receptors incorporating alpha H along with other mouse subunits exhibited a 2-fold higher level of surface alpha-bungarotoxin (BuTx) binding than cells expressing the wild-type mouse AChR. When expressed either alone or with the delta subunit in COS cells, alpha H acquired the BuTx binding conformation (alpha Tx) more efficiently than did alpha M. By oligonucleotide-directed mutagenesis we showed that 2 residues in the amino-terminal domain were responsible for the differences between alpha M and alpha H. Alpha MST, the modified mouse alpha subunit, both folded more efficiently to form alpha Tx and was more effective in forming a stable alpha delta heterodimer than was alpha M. The kinetics of alpha Tx and alpha delta heterodimer formation revealed that the delta subunit increased the conversion of immature forms of the alpha subunit into the BuTx binding form and therefore provides evidence for interaction between the delta subunit and the immature form of the alpha subunit. These results provide evidence of the importance of the amino-terminal domains of the AChR subunits in the assembly process.

Animals↗

PubMed1990

Developmental expression of the Xenopus laevis fos protooncogene.

We have isolated the Xenopus laevis homologue of the protooncogene fos. Nucleotide sequence analysis of the complementary DNA revealed a coding domain for a 370-amino acid protein which shares extensive sequence homology with the avian and mammalian fos protein. During Xenopus development, the fos mRNA is present in the oocyte and egg stages and declines to relatively low levels by the mid-blastula transition. Transient expression of fos can be seen during the late neurula and again at the tadpole stage. The fos mRNA is uniformly distributed in the vegetal and animal poles of the oocytes. In a Xenopus kidney cell line, the fos gene can be transcriptionally activated by serum growth factors and 12-O-tetradecanoylphorbol-13-acetate, and the fos mRNA can be superinduced by cycloheximide. The X. laevis fos protein migrates as a series of bands around Mr 55,000 and is localized in the nucleus. Both the Xenopus fos RNA and fos proteins exhibit a short half-life analogous to that observed with their mammalian counterpart. The Xenopus fos protein readily associates with murine jun (AP-1) oncoprotein, and the complex binds efficiently to AP-1 recognition elements. Furthermore, the Xenopus fos protein collaborates with mammalian jun protein to activate transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive element. The differential expression of fos during Xenopus development and its ability to regulate transcription in association with jun suggest a role during embryogenesis.

Amino Acid Sequence↗