Showing papers on "Protein–protein interaction published in 1990"

System to detect protein-protein interactions

Abstract: Methods are provided for detecting the interaction between a first test protein and a second test protein, in vivo, using reconstitution of the activity of a transcriptional activator. This reconstitution makes use of chimeric genes which express hybrid proteins. Two types of hybrid proteins are prepared. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. If the two test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a marker gene which contains a binding site for the DNA-binding domain.

The U2B'' RNP motif as a site of protein-protein interaction.

Abstract: The U2 snRNP contains two specific proteins, U2B'' and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B'' and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B'' shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1-88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B'' fragment is capable of making specific protein--RNA and protein-protein interactions its structure, as measured by the ability to bind to U2A'', appears to depend on the rest of the protein. Hybrids between U2B'' and the closely related U1A protein are used to localize U2B'' specific amino acids involved in protein-protein interaction. These can be divided into two functional groups. U2A' interaction with U2B'' amino acids 37-46 permits binding to U2 RNA whereas interaction with U2B'' specific amino acids between positions 14 and 25 reduces non-specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein-protein interaction in the assembly of RNPs, particularly since the region of U2'' involved in interaction with U2A' consists mainly of a conserved RNP motif.

Identification of calmodulin-binding proteins.

Abstract: We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Haem peptide–protein interactions. Part 4.—The microperoxidase-8-mediated inhibition of the human placental glutathione S-transferase π catalysed conjugation of glutathione and 1-chloro-2,4-dinitrobenzene

Abstract: The inhibition of the glutathione S-transferase π(GST) catalysed conjugation of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) by the haem octapeptide microperoxidase-8 (MP-8) has been investigated. Incubation of the enzyme with MP-8 results in a pseudo-first-order partial inactivation of the enzyme [kobs= 2.3 (±0.4)× 10–3 s–1; pH 6.5; 22.5 °C]. The kobs is identical to that found for dilutional inactivation of the enzyme observed in buffer alone. MP-8 increases only the extent of inactivation above that observed for dilutional inactivation.Incubation of the enzyme with varying concentrations of MP-8 was followed by a steady-state kinetic study of the enzyme-catalysed conjugation of GSH with CDNB at (a) fixed [GSH], varying [CDNB] and (b) fixed [CDNB] varying [GSH]. In case (a) the data conformed closely with mixed competitive/non-competitive inhibition kinetics (Ki= 3.2 × 10–7 mol dm–3, pH 6.5, 30 °C), while in case (b) the extent of inhibition was insufficient to allow analysis.These observations are rationalized and discussed in the light of the kinetic studies of MP-8 binding to GST π reported in the preceding paper.

From Biological Diversity to Structure-Function Analysis: Protein Engineering in Aspartate Transcarbamoylase

Abstract: Aspartate transcarbamoylase (ATCase, EC 2132) is a common enzyme which catalyzes the first unique step in pyrimidine biosynthesis in divergent biological systems; however, it possesses tremendous architectural variety from one organism to another For example, the E coli ATCase holoenzyme is comprised of two catalytic trimers and three regulatory dimers, while the mammalian enzyme is part of a multifunctional protein aggregate encoding the preceding and subsequent enzymes in pyrimidine biosynthesis Despite extreme differences in quaternary architecture and enzymatic organization, protein engineering studies have demonstrated the existence of highly conserved units of protein structure that impart specific functional characteristics 1 The largest of these units are discrete polypeptides or superdomains within multifunctional proteins which have been shown to be uniquely involved in specific catalytic steps within the CAD or CA complexes of eukaryotic pyrimidine biosynthesis 2 The catalytic polypeptides of various ATCases are organized into two discrete and separable binding domains for its substrates, carbamoyl phosphate and aspartate 3 The regulatory polypeptides of the enteric bacterial enzymes also contain two discrete tertiary domains, the Allosteric Binding Domain and Cys 4 coordinated Zinc Domain involved in the protein:protein interface between the regulatory and catalytic polypeptides of the holoenzyme 4 There are sub-domain structural units within the various polypeptides which have a coordinated impact on specific catalytic and regulatory functions in the enzyme 5 Finally, it has been possible to ascribe some individual function to specific amino acids relative to ligand binding, zinc coordination, protein:protein interactions, and the structural reorganizations in the T-R transition of the enteric holoenzymes

Optical methods for measuring protein-protein interactions

Abstract: The hepatopancreas protein, therefore, probably represents the membranous proton channel of the vacuolar ATPase and electron microscopy has given us the clearest impression yet of its organization; an organization clearly consistent with the functional characteristics of the chloroplast ATPase-CF,, complex [22]. It is hoped that techniques such as electron diffraction may improve the structural resolution obtained. The intriguing question which now arises is the mechanism by which specific unidirectional proton transport is achieved. Clearly, the central channel does not possess a sufficient degree of discrimination. This will most probably be conferred on the system by the additional water-soluble subunits which make up the whole molecular assembly. By analogy with the mitochondria1 ATPase, where DCCD effectively blocks proton transport, it may be that initial uptake by the membrane involves the region between the four helices which constitute the individual subunit. Whatever process occurs, it is clear that the 16 kDa protein presents us with an advantageous system through which the principles of membrane protein structure and assembly can be investigated.

MHV leader RNA secondary structure affects binding to the nucleocapsid protein

Abstract: Leader RNA is found at the 5′ end of the mouse hepatitis virus (MHV) genomic RNA, at the 5′ end of the seven viral mRNAs, and free in the cell. Leader RNA is synthesized by a transcriptional activity separate from the activities that synthesize both (-) sense genomic length RNA and the virus mRNAs1. It is believed to function as a primer, binding to complementary intergenic sites, on (-) sense template, situated 5′ of each of the initiation sites for viral mRNAs2. Using monoclonal antibodies specific for the nucleocapsid (N) protein, immunoprecipitations of RNA/protein complexes from infected cells indicate that the N protein is complexed to: 1) genomic RNA; Z) viral mRNAs; and 3) even free leader containing RNA fragments as small as 60 nucleotides in length3. Northwestern blot analysis showed that the viral N protein exhibits RNA binding activity that is specific for viral leader containing RNA when expressed in the (+) sense4,5. However, this system has several limitations. First, in denaturing conditions, RNA/protein interactions which require the interaction of multiple protein subunits cannot be studied, and second, it is not possible to quantify relative affinities and binding characteristics.

NMR and Molecular Genetics as Tools for Investigating Protein Interactions in Membrane Systems

Abstract: In our laboratory, we have applied the tools of nuclear magnetic resonance (NMR) spectroscopy and molecular genetics to investigate the structural and dynamic properties of membrane-associated proteins and their interactions with membrane components. There are two general classes of membrane proteins, i.e., intrinsic and peripheral ones. For the intrinsic membrane proteins, we have chosen the membranebound D-lactate dehydrogenase (D-LDH) of Escherichia coli as a model to study protein-lipid interactions in membranes. D-LDH is a respiratory enzyme of molecularweight 65, 000 containing flavin adenine dinucleotide (FAD) as a cofactor. The activity of purified D-LDH is enhanced up to 100-fold by lipids and detergents. The gene for D-LDH has been sequenced, and production of the enzyme amplified up to 300-times normal levels. We have biosynthetically incorporated 5-fluorotryptophan (5F-Trp) into D-LDH and studied the five Trp residues by 19F-NMR spectroscopy. In order to gain additional information using 19F-NMR, site-specific, oligonucleotide-directed mutagenesis has been used to insert a sixth Trp into D-LDH at various positions throughout the 571-amino acid chain. These mutant D-LDHs are being characterized biochemically and through NMR. For peripheral membrane proteins, we have chosen two periplasmic binding proteins, histidine-binding protein J (J protein) of Salmonella Typhimurium and glutamine-binding protein (GlnBP) of E. coli as models to investigate the structure-function relationship in periplasmic binding protein-mediated active transport systems. These two proteins both have molecular weights of approximately 25, 000. By using mutant J proteins and GlnBPs and site-specific, oligonucleotide-directed mutagenesis techniques, we have assigned several resonances to specific amino acid residues. We are investigating the relationship between ligand-induced conformational changes in these two proteins and their roles in the active transport of ligand across the cell membrane. We have found that a combination of isotopic labeling, biochemistry, molecular biology, and NMR is a very useful approach to investigate various interactions of membrane-associated protein systems.