scholarly journals Amino Acid Substitutions in Transmembrane Domains 9 and 10 of GerVB That Affect the Germination Properties of Bacillus megaterium Spores

2008 ◽  
Vol 190 (24) ◽  
pp. 8009-8017 ◽  
Author(s):  
Graham Christie ◽  
Christopher R. Lowe
Keyword(s):  
Amino Acid ◽  
Bacillus Megaterium ◽  
Binding Sites ◽  
Molecular Basis ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Receptor Function ◽  
Outer Loop ◽  
Receptor Proteins

ABSTRACT The molecular basis for differences in germinant recognition of Bacillus megaterium QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants. Kinetic analyses of mutants with different amino acid substitutions at these positions indicate that Ser319 and Leu345 are not involved directly in the binding of germinants, but probably reside in regions of the receptor where structural perturbations can affect the conformation of, or access to, germinant binding sites. Position 345 is also shown to be of importance in GerUB, where the F345A mutation severely impairs receptor function. Functionality is restored in the GerUB Ala345 background by substituting putative outer-loop residues adjacent to TM10 for the corresponding residues in GerVB, indicating that a degree of structural coordination between these regions is important to receptor function.

scholarly journals Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

2018 ◽  
Vol 116 (2) ◽  
pp. 679-688 ◽  
Author(s):  
Ming-ling Liao ◽  
George N. Somero ◽  
Yun-wei Dong
Keyword(s):  
Amino Acid ◽  
Thermal Adaptation ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Amino Acid Substitutions ◽  
Thermal Stabilities ◽  
Specific Amino Acid ◽  
Enzyme Thermal Stability ◽  
And Function

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

scholarly journals Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule

1998 ◽  
Vol 331 (2) ◽  
pp. 409-415 ◽  
Author(s):  
Guang-Chao SUI ◽  
Björn WIMAN
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Plasminogen Activator Inhibitor 1 ◽  
Low Activity ◽  
Activator Inhibitor ◽  
Pai 1

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coliand purified by chromatography on heparin–Sepharose and on anhydrotrypsin–agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125 → Lys, Phe126 → Ser and Arg133 → Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125 → Lys and Arg133 → Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t½ 22–31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t½ 2.3 h) as wtPAI-1 (t½ 3.0 h). The mutant Glu130 → Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin–agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.

Phylogeny and Polymorphism in the Long Control Region, E6, and L1 of Human Papillomavirus Types 53, 56, and 66 in Central Brazil

2011 ◽  
Vol 21 (2) ◽  
pp. 222-229 ◽  
Author(s):  
Patrícia Soares Wyant ◽  
Daniela Marreco Cerqueira ◽  
Daniella Sousa Moraes ◽  
José Paulo Gagliardi Leite ◽  
Cláudia Renata Fernandes Martins ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Genetic Variability ◽  
Binding Sites ◽  
Long Control Region ◽  
Amino Acid Substitutions ◽  
Nucleotide Substitutions ◽  
Central Brazil ◽  
Hpv Types ◽  
Genomic Regions

Introduction:Several studies related that different human papillomavirus (HPV) types and intratype variants can present different oncogenic potential. In opposite to HPVs 16 and 18 variants, information about variants of other carcinogenic HPV types is still scarce. The aim of this study was to investigate the genetic variability of HPVs 53, 56, and 66 from Central Brazil isolates.Methods:The long control region (LCR), E6, and L1 genomic regions were amplified and sequenced. We evaluate for nucleotide variations in relation to the reference sequence of each HPV type and also the conservation of physicochemical properties of the deduced amino acid substitutions. In silico analysis was performed to locate binding sites for transcriptional factors within the LCR. Moreover, we performed a phylogenetic analysis with the Central Brazilian and worldwide sequences available at genomic databases.Results:Gathering LCR, E6, and L1 genomic regions, the highest genetic variability was found among HPV-53 isolates with 52 nucleotide variations, followed by HPVs 56 and 66 with 24 and 16 nucleotide substitutions, respectively. The genetic analysis revealed 11 new molecular variants of all HPV types analyzed, totalizing 31 new nucleotide and 3 new amino acid variations. Eight nonconservative amino acid substitutions were detected, which may indicate a biological and pathogenic diversity among HPV types. Furthermore, 8 nucleotide substitutions were localized at putative binding sites for transcription factors in the LCR with a potential implication on viral oncogene expression. The HPVs 53, 56, and 66 phylogenetic analysis confirmed a dichotomic division only described to HPV subtypes and different from the patterns described for HPVs 16 and 18 variants.Conclusions:The high genetic variability observed emphasizes the importance of investigating polymorphisms in types other than HPVs 16 or 18 to better understand the molecular genomic profile of viral infection by different HPV types.

scholarly journals Contribution of Specific Amino Acid Changes in Penicillin Binding Protein 1 to Amoxicillin Resistance in ClinicalHelicobacter pyloriisolates

2010 ◽  
Vol 55 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Nadia N. Qureshi ◽  
Dimitrios Morikis ◽  
Neal L. Schiller
Keyword(s):  
Amino Acid ◽  
Homology Modeling ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Peptic Ulcers ◽  
Penicillin Binding Protein ◽  
Specific Amino Acid ◽  
Binding Cleft ◽  
H Pylori

ABSTRACTAmoxicillin is commonly used to treatHelicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance inH. pyloriis increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4pbp1fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

2001 ◽  
Vol 47 (12) ◽  
pp. 1088-1094 ◽  
Author(s):  
Yew-Loom Chen ◽  
Tsung-Yin Tang ◽  
Kuo-Joan Cheng
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Synergistic Effects ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min–1·(mg protein)–1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

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