Showing papers on "Protein–protein interaction published in 1992"

Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins.

Abstract: The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.

Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.

Abstract: We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS) With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4 Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Abstract: Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.

An alpha-helical hydrophobic hairpin as a specific determinant in protein-protein interaction occurring in Escherichia coli colicin A and B immunity systems.

Abstract: A collection of chimeric pore-forming domains between colicins A and B was constructed to investigate the specific determinants responsible for recognition by the corresponding immunity proteins. The fusion sites in the hybrid proteins were positioned according to the three-dimensional structure of the soluble form of the colicin A pore-forming domain. The hydrophobic hairpin of colicin pore-forming domains, buried in the core of the soluble structure, was the main determinant recognized by the integral immunity proteins. The immunity protein function may require helix-helix recognition within the lipid bilayer.

Myristate-Protein Interactions inPoliovirus: Interactions ofVP4 Threonine 28Contribute totheStructural Conformation of Assembly Intermediates andtheStability ofAssembled Virions

Abstract: The VP4 capsid protein of poliovirus is N-terminally modified with myristic acid. Within the poliovirus structure, a hydrogen bond is observed between the myristate carbonyl and the hydroxyl side chain of threonine 28 of VP4. This interaction is between two fivefold symmetry-related copies of VP4 and is one of several myristoyl-mediated interactions that appears to structurally link the promoters within the pentamer subunit of the virus particle. Site-specific substitutions of the threonine residue were constructed to investigate the biological relevance of these myristate-protein interactions. Replacement of the threonine with glycine or lysine is lethal, generating nonviable viruses. Substitution with serine or valine led to viable viruses, but these mutants displayed anomalies during virus assembly. In addition, both assembled serine- and valine-substituted virion particles showed reduced infectivity and were more sensitive to thermal inactivation and antibody neutralization. Thus the threonine residue provides interactions necessary for efficient assembly of the virus and for virion stability.

The centromere and promoter factor 1 of yeast contains a dimerisation domain located carboxy-terminal to the bHLH domain

Abstract: CPF1 is a basic helix-loop-helix (bHLH) protein required for optimal centromere function and for maintaining methionine independent growth in yeast. In this work, we show that the region carboxy-terminal to the bHLH domain of CPF1 is essential for CPF1 function in the cell and for dimerisation of CPF1 in solution. The C-terminus of CPF1 contains a potential long amphipathic helix with a hydrophobic face which could provide a suitable protein:protein interface. Point mutations in residues forming this hydrophobic face are sufficient to weaken the interaction between the protein and DNA. By fusing the DNA binding domain or the transcriptional activation domain of GAL4 to the C-terminal 87 amino acids of CPF1, we show that this region is sufficient for mediating protein:protein interactions in vivo. The C-terminal domain of CPF1 can be replaced by the leucine repeat region of the bHLH-ZIP protein USF and the hybrid CPF1-USF protein functions in vivo to provide normal centromere function and methionine independent growth. However, the CPF1-USF hybrid protein is unable to interact with CPF1 suggesting that a dimer of CPF1 is sufficient for maintaining methionine independent growth and normal centromere function.

Molecular analysis of the protein‐protein interaction between the E9 immunity protein and colicin E9

Abstract: The specificity-determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16-43 of the 86-amino-acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity-determining residues. Using site-directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity-determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low-level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity-determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5'-dithiobis(2-nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti-DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity-determining region.

Protein-protein interaction in the α-complementation system of β-galactosidase

Abstract: Publisher Summary This chapter discusses protein–protein interaction in the α-complementation system of β-galactosidase. α-complementation system in β-galactosidase system is a system of protein–protein interaction where active enzyme is formed through the association of two different polypeptide chains encoded by different lacZ alleles. The chapter presents some of the key features of α-complementation. β-galactosidase is a tetrameric enzyme. The rate-determining step in α-complementation is the conversion of inactive dimeric proteins, termed α-acceptors, to catalytically active tetramers. The most thoroughly studied α-donor analyzed in vitro is a cyanogen bromide peptide derived from β-galactosidase, comprising amino acid residues 3–92 of the wild-type protein. Either the M15 protein, lacking residues 11–41, or the M112 protein, with a smaller deletion of residues 23–31, are able to serve as α-acceptors. Immunochemical analyses and amino acid sequence comparisons of the α-donor and α-acceptor molecules suggest that residues 23–31 of β-galactosidase play a critical role in the dimerization of M15 dimers.