High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: application to study structure-function of plasminogen activation inhibitor 1 (PAI-I)

2000 ◽  
Vol 301 (5) ◽  
pp. 1135-1147 ◽  
Author(s):  
A.Allart Stoop ◽  
Laurent Jespers ◽  
Ignace Lasters ◽  
Eric Eldering ◽  
Hans Pannekoek
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Structure Function ◽  
Protein Interactions ◽  
Essential Amino Acid ◽  
High Density ◽  
Dna Shuffling ◽  
Plasminogen Activation ◽  
Amino Acid Residues ◽  
Protein Protein Interactions

scholarly journals Peptide recognition and dephosphorylation by the vaccinia VH1 phosphatase

2020 ◽  
Author(s):  
Bryan M. Zhao ◽  
Megan Hogan ◽  
Michael S Lee ◽  
Beverly K. Dyas ◽  
Robert G. Ulrich
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Host Cell Proteins ◽  
Enzyme Substrate ◽  
Dual Specificity ◽  
Contact Residues

ABSTRACTThe VH1 protein encoded by the highly conserved H1 locus of orthopoxviruses is a dual-specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are required for virus replication, VH1 is a prime target for the development of therapeutic inhibitors. However, the presentation of a shallow catalytic site has thwarted all drug development efforts. As an alternative to direct targeting of catalytic pockets, we describe surface contacts between VH1 and substrates that are essential for full activity and provide a new pathway for developing inhibitors of protein-protein interactions. Critical amino acid residues were manipulated by site-directed mutagenesis of VH1, and perturbation of peptide substrate interactions based on these mutations were assessed by high-throughput assays that employed surface plasmon resonance and phosphatase activities. Two positively-charged residues (Lys-20 and Lys-22) and the hydrophobic side chain of Met-60 appear to orient the polarity of the pTyr peptide on the VH1 surface, while additional amino acid residues that flank the catalytic site contribute to substrate recognition and productive dephosphorylation. We propose that the enzyme-substrate contact residues described here may serve as molecular targets for the development of inhibitors that specifically block VH1 catalytic activity and thus poxvirus replication.

Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

2004 ◽  
Vol 36 (2) ◽  
pp. 93-98 ◽  
Author(s):  
Gang Liu ◽  
Guang-Xiu Lu ◽  
Xiao-Wei Xing
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Critical Role ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Spermatogenic Cells ◽  
Protein Protein Interactions ◽  
New Gene ◽  
Pcr Technology ◽  
Dnak Protein

Abstract Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

scholarly journals Identification of amino acid residues required for Ras p21 target activation.

1991 ◽  
Vol 11 (8) ◽  
pp. 3997-4004 ◽  
Author(s):  
M S Marshall ◽  
L J Davis ◽  
R D Keys ◽  
S D Mosser ◽  
W S Hill ◽  
...  
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Protein Interactions ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Protein Protein Interactions ◽  
Biological Assays ◽  
Ras P21

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

A structural view of the dissociation ofEscherichia colitryptophanase

2015 ◽  
Vol 71 (12) ◽  
pp. 2364-2371
Author(s):  
Keren Green ◽  
Nasrin Qasim ◽  
Garik Gdaelvsky ◽  
Anna Kogan ◽  
Yehuda Goldgur ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Hydrophobic Interactions ◽  
Molecular Axis ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
E Coli ◽  
Reversible Loss ◽  
Structure Of Proteins

Tryptophanase (Trpase) is a pyridoxal 5′-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer.Escherichia coliTrpase dissociates into dimers, whileProteus vulgarisTrpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein–protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between theE. coliandP. vulgarisTrpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of theE. colienzyme were created to resemble the amino-acid sequence ofP. vulgarisTrpase. In one group, residues 15 and 59 that are located along the molecular axisR(also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axisQ(also termed the catalytic axis). Replacing amino-acid residues along theRaxis resulted in dissociation of the tetramers into monomers, similar to theP. vulgarisTrpase, while replacing amino-acid residues along theQaxis resulted in dissociation into dimers only. The crystal structure of the V59M mutant ofE. coliTrpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that inE. coliTrpase hydrophobic interactions along theRaxis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along theQaxis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation ofE. coliTrpase into dimers occurs along the molecularQaxis.

Identification of amino acid residues required for Ras p21 target activation

1991 ◽  
Vol 11 (8) ◽  
pp. 3997-4004
Author(s):  
M S Marshall ◽  
L J Davis ◽  
R D Keys ◽  
S D Mosser ◽  
W S Hill ◽  
...  
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Protein Interactions ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Protein Protein Interactions ◽  
Biological Assays ◽  
Ras P21

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

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