Pub Date : 2023-08-31DOI: 10.1007/s10930-023-10137-1
Magnus Lundborg, Jack Lidmar, Berk Hess
Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.
{"title":"On the Path to Optimal Alchemistry","authors":"Magnus Lundborg, Jack Lidmar, Berk Hess","doi":"10.1007/s10930-023-10137-1","DOIUrl":"10.1007/s10930-023-10137-1","url":null,"abstract":"<div><p>Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10930-023-10137-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4048271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-08-27DOI: 10.1007/s10930-023-10145-1
Mujib Abdulkadir Abdurahman, İnci Durukan, Tuba Dinçer, Serap Pektaş, Ersin Karataş, Ali Osman Kiliç
Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.
噬菌体内溶素作为一种新型抗菌剂,在食品保鲜方面具有广阔的应用前景。在本研究中,来自金黄色葡萄球菌亚群的内溶素。对金黄色葡萄球菌ATCC 27692-B1噬菌体52 (LysSA52)进行了克隆、表达和抑菌鉴定。从噬菌体52中提取DNA,通过PCR扩增得到含有内溶素基因(lysSA52)的1446 bp DNA片段,并将其克隆到pET SUMO表达载体中。阳性克隆经测序和开放阅读框分析证实。LysSA52序列与葡萄球菌噬菌体SA12、SA13和DSW2等噬菌体的内溶酶具有高度同源性。克隆的lysSA52基因编码481个氨基酸的内溶素,在大肠杆菌BL21中表达,计算分子量为66 kDa (lysSA52)。重组内溶素LysSA52对10种革兰氏阳性细菌中的8种表现出裂解活性,包括耐甲氧西林金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌、肺炎链球菌、化脓性链球菌、粪肠球菌、粪肠球菌和萎缩芽孢杆菌。此外,0.50 mg/mL的LysSA52内溶素处理12 h后,在微滴板上建立的金黄色葡萄球菌和表皮葡萄球菌生物膜减少了约60%。本研究的数据表明,无论葡萄球菌和其他几种革兰氏阳性致病菌的耐药性如何,LysSA52内溶素都可以作为一种抗菌蛋白来预防和治疗其引起的感染。
{"title":"Staphylococcus aureus Bacteriophage 52 Endolysin Exhibits Anti-Biofilm and Broad Antibacterial Activity Against Gram-Positive Bacteria","authors":"Mujib Abdulkadir Abdurahman, İnci Durukan, Tuba Dinçer, Serap Pektaş, Ersin Karataş, Ali Osman Kiliç","doi":"10.1007/s10930-023-10145-1","DOIUrl":"10.1007/s10930-023-10145-1","url":null,"abstract":"<div><p>Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from <i>Staphylococcus aureus</i> subsp. <i>aureus</i> ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (<i>lysSA52</i>) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned <i>lys</i>SA52 gene encoding 481 amino acids endolysin was expressed in <i>Escherichia coli</i> BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant <i>Staphylococcus aureus</i>, <i>Staphylococcus epidermidis</i>, <i>Staphylococcus haemolyticus</i>, <i>Streptococcus pneumonia</i>, <i>Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus.</i> In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of <i>S. aureus</i> and <i>S. epidermidis</i> established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5032064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes.
Method
In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties.
Result
Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase’s asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme.
Conclusion
In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.
{"title":"Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis","authors":"Mahdieh Mahboobi, Ali-Hatef Salmanian, Hamid Sedighian, Bijan Bambai","doi":"10.1007/s10930-023-10149-x","DOIUrl":"10.1007/s10930-023-10149-x","url":null,"abstract":"<div><h3>Introduction</h3><p>L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes.</p><h3>Method</h3><p>In this study, firstly to find the appropriate mutation on glycine 88, various <i>in silico</i> analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties.</p><h3>Result</h3><p>Our <i>in silico</i> findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase’s asparaginase activity. The catalytic efficiency of each enzyme (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the G88Q mutant is 36.32% higher than the <i>Escherichia coli</i> K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (<i>k</i><sub>cat</sub>) with ASN as substrate relative to the wild type enzyme.</p><h3>Conclusion</h3><p><i>In silico analyses</i> and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10083893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-08-27DOI: 10.1007/s10930-023-10138-0
Nassim Faridi, Maryam Sanjari-Pour, Ping Wang, S. Zahra Bathaie
The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1–42 (Aβ1−42) peptide is very hydrophobic and quickly forms the β-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aβ1−42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aβ1−42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aβ1−42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aβ1−42 at low concentration (25 µM) in Ham’s/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aβ1−42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aβ1−42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aβ1−42 aggregates with a particular size and molecular structure.
{"title":"The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aβ1−42 at Different Concentrations","authors":"Nassim Faridi, Maryam Sanjari-Pour, Ping Wang, S. Zahra Bathaie","doi":"10.1007/s10930-023-10138-0","DOIUrl":"10.1007/s10930-023-10138-0","url":null,"abstract":"<div><p>The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1–42 (Aβ<sub>1−42</sub>) peptide is very hydrophobic and quickly forms the β-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aβ<sub>1−42</sub> peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aβ<sub>1−42</sub> structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aβ<sub>1−42</sub> fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aβ<sub>1−42</sub> at low concentration (25 µM) in Ham’s/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aβ<sub>1−42</sub> at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aβ<sub>1−42</sub> oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aβ<sub>1−42</sub> aggregates with a particular size and molecular structure.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10930-023-10138-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5032078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-08-25DOI: 10.1007/s10930-023-10151-3
Chaofei Cheng, Zhendong Zhao, Guangzhi Liu
γδ T cells, especially Vγ9Vδ2 T cells, play an important role in mycobacterial infection. We have identified some Vγ9Vδ2 T cells that recognize protein/peptide antigens derived from mycobacteria, which may induce protective immune responses to mycobacterial infection. To clarify the structural basis of the molecular recognition mechanism, we tried many methods to express the Vγ9Vδ2 T-cell receptor (TCR). The Vγ9Vδ2 TCR was not expressed well in a prokaryotic expression system or a baculovirus expression system, even after extensive optimization. In a mammalian cell expression system, the Vγ9Vδ2 TCR was expressed in the form of a soluble heterodimer, which was suitable for crystal screening. Reduced-temperature cultivation (cold shock) increased the yield of the recombinant TCR. The recombinant purified TCR was used for crystal trials, and crystals that could be used for X-ray diffraction were obtained. Although we have not yet determined the crystal structure of the Vγ9Vδ2 TCR, we have established a procedure for Vγ9Vδ2 TCR expression and purification, which is useful for basic research and potentially for clinical application.
{"title":"Expression, Purification, and Crystallization of the Vγ9Vδ2 T-cell Receptor Recognizing Protein/Peptide Antigens","authors":"Chaofei Cheng, Zhendong Zhao, Guangzhi Liu","doi":"10.1007/s10930-023-10151-3","DOIUrl":"10.1007/s10930-023-10151-3","url":null,"abstract":"<div><p>γδ T cells, especially Vγ9Vδ2 T cells, play an important role in mycobacterial infection. We have identified some Vγ9Vδ2 T cells that recognize protein/peptide antigens derived from mycobacteria, which may induce protective immune responses to mycobacterial infection. To clarify the structural basis of the molecular recognition mechanism, we tried many methods to express the Vγ9Vδ2 T-cell receptor (TCR). The Vγ9Vδ2 TCR was not expressed well in a prokaryotic expression system or a baculovirus expression system, even after extensive optimization. In a mammalian cell expression system, the Vγ9Vδ2 TCR was expressed in the form of a soluble heterodimer, which was suitable for crystal screening. Reduced-temperature cultivation (cold shock) increased the yield of the recombinant TCR. The recombinant purified TCR was used for crystal trials, and crystals that could be used for X-ray diffraction were obtained. Although we have not yet determined the crystal structure of the Vγ9Vδ2 TCR, we have established a procedure for Vγ9Vδ2 TCR expression and purification, which is useful for basic research and potentially for clinical application.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2023-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10930-023-10151-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10058071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-08-25DOI: 10.1007/s10930-023-10141-5
Pavel Buslaev, Gerrit Groenhof
Molecular dynamics (MD) simulations are routinely performed of biomolecules in solution, because this is their native environment. However, the structures used in such simulations are often obtained with X-ray crystallography, which provides the atomic coordinates of the biomolecule in a crystal environment. With the advent of free electron lasers and time-resolved techniques, X-ray crystallography can now also access metastable states that are intermediates in a biochemical process. Such experiments provide additional data, which can be used, for example, to optimize MD force fields. Doing so requires that the simulation of the biomolecule is also performed in the crystal environment. However, in contrast to simulations of biomolecules in solution, setting up a crystal is challenging. In particular, because not all solvent molecules are resolved in X-ray crystallography, adding a suitable number of solvent molecules, such that the properties of the crystallographic unit cell are preserved in the simulation, can be difficult and typically is a trial-and-error based procedure requiring manual interventions. Such interventions preclude high throughput applications. To overcome this bottleneck, we introduce gmXtal, a tool for setting up crystal simulations for MD simulations with GROMACS. With the information from the protein data bank (rcsb.org) gmXtal automatically (i) builds the crystallographic unit cell; (ii) sets the protonation of titratable residues; (iii) builds missing residues that were not resolved experimentally; and (iv) adds an appropriate number of solvent molecules to the system. gmXtal is available as a standalone tool https://gitlab.com/pbuslaev/gmxtal.