scholarly journals Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

2015 ◽  
Vol 112 (30) ◽  
pp. 9328-9333 ◽  
Author(s):  
Colin A. Kretz ◽  
Manhong Dai ◽  
Onuralp Soylemez ◽  
Andrew Yee ◽  
Karl C. Desch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzyme Kinetics ◽  
High Throughput Sequencing ◽  
Phage Display Library ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Reaction Rate Constants ◽  
Binding Domains ◽  
Dependent Change ◽  
Library Complexity

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

scholarly journals Massively parallel single-amino-acid mutagenesis

2015 ◽  
Vol 12 (3) ◽  
pp. 203-206 ◽  
Author(s):  
Jacob O Kitzman ◽  
Lea M Starita ◽  
Russell S Lo ◽  
Stanley Fields ◽  
Jay Shendure
Keyword(s):  
Amino Acid ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Amino Acid Mutagenesis

scholarly journals Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

2018 ◽  
Author(s):  
Kenneth A. Matreyek ◽  
Lea M. Starita ◽  
Jason J. Stephany ◽  
Beth Martin ◽  
Melissa A. Chiasson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Massively Parallel Sequencing ◽  
Dominant Negative ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Protein Variant ◽  
Parallel Sequencing ◽  
Missense Variants ◽  
Functional Variants ◽  
Amino Acid Variants

ABSTRACTDetermining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. We identify 1,079 PTEN and 805 TPMT single amino acid variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and our abundance data suggest that a PTEN variant accounting for ~10% of PTEN missense variants in melanomas functions via a dominant negative mechanism. Finally, we demonstrate that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable assay for characterizing missense variants.

scholarly journals Feline Leukemia Virus Envelope Sequences That Affect T-Cell Tropism and Syncytium Formation Are Not Part of Known Receptor-Binding Domains

2000 ◽  
Vol 74 (13) ◽  
pp. 5754-5761 ◽  
Author(s):  
Samuel R. Gwynn ◽  
F. Claire Hankenson ◽  
Adam S. Lauring ◽  
Jennifer L. Rohn ◽  
Julie Overbaugh
Keyword(s):  
Tissue Culture ◽  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Envelope Protein ◽  
Leukemia Virus ◽  
Feline Leukemia Virus ◽  
Single Amino Acid ◽  
Binding Domains ◽  
Amino Acid Insertion

ABSTRACT The envelope protein is a primary pathogenic determinant for T-cell-tropic feline leukemia virus (FeLV) variants, the best studied of which is the immunodeficiency-inducing virus, 61C. We have previously demonstrated that T-cell-tropic, cytopathic, and syncytium-inducing viruses evolve in cats infected with a relatively avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T variant, which encoded scattered single-amino-acid changes throughout the envelope as well as a 4-amino-acid insertion in the C-terminal half of the surface unit (SU) of envelope, was sufficient to confer the T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser, S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol. 72:2686–2696, 1998). In the present study, we examined the role of the 4-amino-acid insertion in determining viral replication and tropism of FeLV-81T. The 4-amino-acid insertion was found to be functionally equivalent to a 6-amino-acid insertion at an identical location in the 61C variant. However, viruses expressing a chimeric 61E/81T SU, containing the insertion together with the N terminus of 61E SU, were found to be replication defective and were impaired in the processing of the envelope precursor into the functional SU and transmembrane (TM) proteins. In approximately 10% of cultured feline T cells (3201) transfected with the 61E/81T envelope chimeras and maintained over time, replication-competent tissue culture-adapted variants were isolated. Compensatory mutations in the SU of the tissue culture-adapted viruses were identified at positions 7 and 375, and each was shown to restore envelope protein processing when combined with the C-terminal 81T insertion. Unexpectedly, these viruses displayed different phenotypes in feline T cells: the virus with a change from glutamine to proline at position 7 acquired a T-cell-tropic, cytopathic phenotype, whereas the virus with a change from valine to leucine at position 375 had slower replication kinetics and caused no cytopathic effects. Given the differences in the replication properties of these viruses, it is noteworthy that the insertion as well as the two single-amino-acid changes all occur outside of predicted FeLV receptor-binding domains.

scholarly journals Erratum: Corrigendum: Massively parallel single-amino-acid mutagenesis

2017 ◽  
Vol 14 (5) ◽  
pp. 540-540
Author(s):  
Jacob O Kitzman ◽  
Lea M Starita ◽  
Russell S Lo ◽  
Stanley Fields ◽  
Jay Shendure
Keyword(s):  
Amino Acid ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Amino Acid Mutagenesis

scholarly journals Amino acid torsion angles enable prediction of protein fold classification

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kun Tian ◽  
Xin Zhao ◽  
Xiaogeng Wan ◽  
Stephen S.-T. Yau
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Protein Structure Prediction ◽  
Structure Prediction ◽  
High Throughput Sequencing ◽  
Protein Structures ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
New Approach ◽  
Protein Functions

AbstractProtein structure can provide insights that help biologists to predict and understand protein functions and interactions. However, the number of known protein structures has not kept pace with the number of protein sequences determined by high-throughput sequencing. Current techniques used to determine the structure of proteins are complex and require a lot of time to analyze the experimental results, especially for large protein molecules. The limitations of these methods have motivated us to create a new approach for protein structure prediction. Here we describe a new approach to predict of protein structures and structure classes from amino acid sequences. Our prediction model performs well in comparison with previous methods when applied to the structural classification of two CATH datasets with more than 5000 protein domains. The average accuracy is 92.5% for structure classification, which is higher than that of previous research. We also used our model to predict four known protein structures with a single amino acid sequence, while many other existing methods could only obtain one possible structure for a given sequence. The results show that our method provides a new effective and reliable tool for protein structure prediction research.

The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution

FEBS Letters ◽  
1995 ◽  
Vol 372 (1) ◽  
pp. 96-98 ◽  
Author(s):  
Markus Linder ◽  
Gunnar Lindeberg ◽  
Tapani Reinikainen ◽  
Tuula T. Teeri ◽  
Göran Pettersson
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Binding Domains ◽  
The Difference ◽  
Cellulose Binding

Single amino acid exchanges in FAD-binding domains of squalene epoxidase of Saccharomyces cerevisiae lead to either loss of functionality or terbinafine sensitivity

2005 ◽  
Vol 33 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
C. Ruckenstuhl ◽  
A. Eidenberger ◽  
S. Lang ◽  
F. Turnowsky
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Binding Domains ◽  
Essential Enzyme ◽  
Fad Binding ◽  
Acid Exchange

Squalene epoxidase (Erg1p) is an essential enzyme in the ergosterol biosynthesis pathway in yeast. For its enzymatic activity, Erg1p requires molecular oxygen, NAD(P)H and FAD. Amino acid analysis and sequence alignment with other squalene epoxidases revealed two highly conserved FAD-binding domains, FAD I and FAD II. By random PCR mutagenesis of the ERG1 gene, one erg1 allele was isolated that carries a mutation leading to a single amino acid exchange in the FAD I domain close to the N-terminus of Erg1p. This erg1 allele codes for functional squalene epoxidase and renders yeast cells hypersensitive to terbinafine. Amino acid exchanges of other conserved residues in the FAD I and FAD II regions either led to non-functional squalene epoxidase or to the formation of squalene epoxidase with wild-type properties. These results describe the importance of specific amino acids for enzymatic activity in the yeast squalene epoxidase Erg1p.

scholarly journals Binding and Regulation of Transcription by Yeast Ste12 Variants To Drive Mating and Invasion Phenotypes

Genetics ◽  
2019 ◽  
Vol 214 (2) ◽  
pp. 397-407 ◽  
Author(s):  
Wei Zhou ◽  
Michael W. Dorrity ◽  
Kerry L. Bubb ◽  
Christine Queitsch ◽  
Stanley Fields
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Cellular Response ◽  
Expression Profiles ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Regulation Of Transcription ◽  
Coding Region ◽  
Binding Domains

Amino acid substitutions are commonly found in human transcription factors, yet the functional consequences of much of this variation remain unknown, even in well-characterized DNA-binding domains. Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12—a yeast transcription factor regulating mating and invasion—alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the “calling-card” method, RNA sequencing, and HT-SELEX (high throughput systematic evolution of ligands by exponential enrichment), we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions.

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