scholarly journals The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3858
Author(s):  
Nurul Nadirah Ahmad ◽  
Nor Hafizah Ahmad Kamarudin ◽  
Adam Thean Chor Leow ◽  
Raja Noor Zaliha Raja Abd. Rahman
Keyword(s):  
Double Mutant ◽  
Enzyme Stability ◽  
Conformational Stability ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Relative Activity ◽  
Wild Type ◽  
Single Mutant ◽  
Lysine Residues ◽  
The Stability

Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.

scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Double mutant of chymotrypsin inhibitor 2 stabilized through increased conformational entropy

2021 ◽  
Author(s):  
Yulian Gavrilov ◽  
Felix Kümmerer ◽  
Simone Orioli ◽  
Andreas Prestel ◽  
Kresten Lindorff-Larsen ◽  
...  
Keyword(s):  
Double Mutant ◽  
Conformational Dynamics ◽  
Nmr Relaxation ◽  
Conformational Heterogeneity ◽  
Native State ◽  
Chymotrypsin Inhibitor 2 ◽  
Relaxation Measurements ◽  
The Stability ◽  
Dynamics Simulations ◽  
Mutant Forms

The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side-chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: A reduction in conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

The reactivation of tabun-inhibited mutant AChE with Ortho-7: steered molecular dynamics and quantum chemical studies

2016 ◽  
Vol 12 (4) ◽  
pp. 1224-1231 ◽  
Author(s):  
Rabindranath Lo ◽  
Nellore Bhanu Chandar ◽  
Shibaji Ghosh ◽  
Bishwajit Ganguly
Keyword(s):  
Molecular Dynamics ◽  
In Silico ◽  
Quantum Chemical ◽  
Double Mutant ◽  
Steered Molecular Dynamics ◽  
Wild Type ◽  
Single Mutant ◽  
Chemical Studies ◽  
Quantum Chemical Studies

Tabun inhibited AChE can be reactivated more easily with a single mutant than with a wild-type or double mutant: an in silico study.

scholarly journals Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2445-2455 ◽  
Author(s):  
Bethany A. Rader ◽  
Christopher Wreden ◽  
Kevin G. Hicks ◽  
Emily Goers Sweeney ◽  
Karen M. Ottemann ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Double Mutant ◽  
Soft Agar ◽  
Dependent Manner ◽  
Wild Type ◽  
Environmental Chemical ◽  
Single Mutant ◽  
Autoinducer 2 ◽  
Reduced Frequency ◽  
H Pylori

Helicobacter pylori moves in response to environmental chemical cues using a chemotaxis two-component signal-transduction system. Autoinducer-2 (AI-2) is a quorum-sensing signal produced by the LuxS protein that accumulates in the bacterial environment in a density-dependent manner. We showed previously that a H. pylori luxS mutant was defective in motility on soft agar plates. Here we report that deletion of the luxS gene resulted in swimming behaviour with a reduced frequency of stops as compared to the wild-type strain. Stopping frequency was restored to wild-type levels by genetic complementation of the luxS mutation or by addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Synthetic DPD also increased the frequency of stops in wild-type H. pylori, similar to the behaviour induced by the known chemorepellent HCl. We found that whereas mutants lacking the chemoreceptor genes tlpA, tlpC or tlpD responded to an exogenous source of synthetic DPD, the chemoreceptor mutant tlpB was non-responsive to a gradient or uniform distribution of the chemical. Furthermore, a double mutant lacking both tlpB and luxS exhibited chemotactic behaviour similar to the tlpB single mutant, whereas a double mutant lacking both tlpB and the chemotransduction gene cheA behaved like a nonchemotactic cheA single mutant, supporting the model that tlpB functions in a signalling pathway downstream of luxS and upstream of cheA. We conclude that H. pylori perceives LuxS-produced AI-2 as a chemorepellent via the chemoreceptor TlpB.

scholarly journals Rational Protein Engineering to Increase the Activity and Stability of IsPETase Using the PROSS Algorithm

Polymers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 3884
Author(s):  
Andrew Rennison ◽  
Jakob R. Winther ◽  
Cristiano Varrone
Keyword(s):  
Thermal Stability ◽  
Enzyme Stability ◽  
Fold Increase ◽  
Wild Type ◽  
Bioinformatic Tools ◽  
Mild Conditions ◽  
Degradation Activity ◽  
Packaging Industry ◽  
The Stability ◽  
Thermal Stability Of

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

scholarly journals Enhancing ubiquitin crystallization through surface-entropy reduction

2014 ◽  
Vol 70 (10) ◽  
pp. 1434-1442 ◽  
Author(s):  
Patrick J. Loll ◽  
Peining Xu ◽  
John T. Schmidt ◽  
Scott L. Melideo
Keyword(s):  
Surface Density ◽  
Double Mutant ◽  
High Surface ◽  
Wild Type ◽  
Mutant Proteins ◽  
X Ray ◽  
Surface Entropy ◽  
Lysine Residues ◽  
Entropy Reduction ◽  
Packing Interactions

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

scholarly journals An msbB Homologue Carried in Plasmid pO157 Encodes an Acyltransferase Involved in Lipid A Biosynthesis in Escherichia coli O157:H7

2004 ◽  
Vol 72 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Russell E. Bishop ◽  
Carlton Gyles
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Double Mutant ◽  
Lipid A ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
Single Mutant ◽  
Double Mutant Strain ◽  
Mutant Strains ◽  
Lipid A Biosynthesis

ABSTRACT Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.

scholarly journals Separate Ion Pathways in a Cl−/H+ Exchanger

2005 ◽  
Vol 126 (6) ◽  
pp. 563-570 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Walden ◽  
Wang Nguitragool ◽  
Hariharan Jayaram ◽  
Carole Williams ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Transport Mechanism ◽  
Double Mutant ◽  
Wild Type ◽  
Extracellular Solution ◽  
X Ray ◽  
Single Mutant ◽  
Cl Transport ◽  
Alternating Access

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

scholarly journals Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication

2006 ◽  
Vol 87 (8) ◽  
pp. 2397-2401 ◽  
Author(s):  
Koki Fujisaki ◽  
Gerald B. Ravelo ◽  
Satoshi Naito ◽  
Masayuki Ishikawa
Keyword(s):  
Arabidopsis Thaliana ◽  
Coat Protein ◽  
Double Mutant ◽  
Detectable Level ◽  
Expression Level ◽  
Wild Type ◽  
Homologous Proteins ◽  
Single Mutant ◽  
Mutant Lines

The TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.

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