Thermal Stability of Proteins

ABSTRACT
Marine natural compounds are required by industries to improve and reduce costs of many biotechnological processes. In this work, specific activity of midgut gland digestive enzymes was measured in seven species of decapods. Alkaline and acid peptidase, lipase and amylase activities were registered in Pleoticus muelleri (Pm), Artemesia longinaris (Al), Paralomis granulosa (Pg), Lithodes santolla (Ls), Munida gregaria (Mg), Neohelice granulata (Ng), and Ovalipes trimaculatus (Ot). The highest activities for all the analyzed digestive enzymes were obtained in Pg, Ls, Mg and Ot, which were used for optimum temperature and pH measurements and to test their stability at different temperatures and pH. In general, optimal enzyme temperatures were between 30 and 50 °C in the four crustaceans. Peptidases and lipases from these species showed in general alkaline optimal pH except for those from Ot which presented high activity at the whole pH range (2-12). Amylase from Ot presented its optimal at pH 6-8 and 30-50 °C. Thermal stability of peptidases was elevated at 30°C for the four species tested (Pg; Ls; Mg and Ot), lipases were stable mainly at 30-50 °C and amylase was stable at 10°C, 30°C and 50°C during the first 30 minutes of the assay. In general, stability of the four enzymes from Pg was greater at alkaline pH than at acid. The other species showed stability at all pH for the four enzymes. The range of temperature and pH of the tested enzymes activity are coincident with those used in various biotechnological processes and therefore indicates the potential of these enzymes to be used for industrial procedures. Further, most of the species studied are converted into fishery residues, either by processing or by-catch, so their reuse would also contribute to the sustainability of the activity.

Keywords: biochemistry, biotechnology, Crustacea, digestive enzymes, marine compounds

b-lactamases are enzymes that deactivate b-lactam antibiotics through a hydrolysis mechanism. There are two known types of b-lactamases: serine b-lactamases (SBLs) and metallo b-lactamases (MBLs). The two existing strategies to overcome b-lactamase-mediated resistance are (a) to develop novel b-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop b-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (#2 mg L −1) against New Delhi metallo-b-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available b-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (K i = 0.85 and 1.87, respectively). This study has revealed a novel series of b-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs. cantly to antibiotic resistance. 1,3,4 The Antimicrobial Resistance (AMR) National Strategy Framework (2018-2024), as well as the Global Research and Development priority setting for AMR, and the Global Antibiotic Research and Development Partnership, prioritise research regarding antimicrobial treatment to help prevent obsolete antibiotics emanating as a result of mutations and bacterial evolution. 5-7 Antibiotic resistance has worsened, due to the empirical treatment of hospitalised COVID-19 patients. 3,8,9 There are several initiatives in place to either raise awareness, help reduce, or keep track of resistance, such as: the Global Action Plan on Antimicrobial Resistance (GAP); World Antimicrobial Awareness Week (WAAW); The Global Antimicrobial Resistance and Use Surveillance System (GLASS); Global Research and Development priority setting for AMR; Access Watch Reserve (AWaRe); and Global Antibiotic Research and Development Partnership (GARDP). 7 Murray et al. estimated that 4.95 million deaths occurred in the year 2019 as a result of antibiotic resistance. 6 Identied Klebsiella pneumoniae, as a common pathogen accounting for 29% of all reported bacterial infections. 7 Klebsiella pneumoniae has recently been identied as a bacteria of concern, as mentioned in several reports, studies, and reviews, and is further substantiated by the resistance map (Fig. 1) generated from the CDDEP (Centre for Disease Dynamics, Economics and Policy). 10 Unfortunately,

b-lactamases are enzymes that deactivate b-lactam antibiotics through a hydrolysis mechanism. There are two known types of b-lactamases: serine b-lactamases (SBLs) and metallo b-lactamases (MBLs). The two existing strategies to overcome b-lactamase-mediated resistance are (a) to develop novel b-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop b-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (#2 mg L −1) against New Delhi metallo-b-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available b-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (K i = 0.85 and 1.87, respectively). This study has revealed a novel series of b-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs. cantly to antibiotic resistance. 1,3,4 The Antimicrobial Resistance (AMR) National Strategy Framework (2018-2024), as well as the Global Research and Development priority setting for AMR, and the Global Antibiotic Research and Development Partnership, prioritise research regarding antimicrobial treatment to help prevent obsolete antibiotics emanating as a result of mutations and bacterial evolution. 5-7 Antibiotic resistance has worsened, due to the empirical treatment of hospitalised COVID-19 patients. 3,8,9 There are several initiatives in place to either raise awareness, help reduce, or keep track of resistance, such as: the Global Action Plan on Antimicrobial Resistance (GAP); World Antimicrobial Awareness Week (WAAW); The Global Antimicrobial Resistance and Use Surveillance System (GLASS); Global Research and Development priority setting for AMR; Access Watch Reserve (AWaRe); and Global Antibiotic Research and Development Partnership (GARDP). 7 Murray et al. estimated that 4.95 million deaths occurred in the year 2019 as a result of antibiotic resistance. 6 Identied Klebsiella pneumoniae, as a common pathogen accounting for 29% of all reported bacterial infections. 7 Klebsiella pneumoniae has recently been identied as a bacteria of concern, as mentioned in several reports, studies, and reviews, and is further substantiated by the resistance map (Fig. 1) generated from the CDDEP (Centre for Disease Dynamics, Economics and Policy). 10 Unfortunately,

b-lactamases are enzymes that deactivate b-lactam antibiotics through a hydrolysis mechanism. There are two known types of b-lactamases: serine b-lactamases (SBLs) and metallo b-lactamases (MBLs). The two existing strategies to overcome b-lactamase-mediated resistance are (a) to develop novel b-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop b-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (#2 mg L −1) against New Delhi metallo-b-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available b-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (K i = 0.85 and 1.87, respectively). This study has revealed a novel series of b-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs. cantly to antibiotic resistance. 1,3,4 The Antimicrobial Resistance (AMR) National Strategy Framework (2018-2024), as well as the Global Research and Development priority setting for AMR, and the Global Antibiotic Research and Development Partnership, prioritise research regarding antimicrobial treatment to help prevent obsolete antibiotics emanating as a result of mutations and bacterial evolution. 5-7 Antibiotic resistance has worsened, due to the empirical treatment of hospitalised COVID-19 patients. 3,8,9 There are several initiatives in place to either raise awareness, help reduce, or keep track of resistance, such as: the Global Action Plan on Antimicrobial Resistance (GAP); World Antimicrobial Awareness Week (WAAW); The Global Antimicrobial Resistance and Use Surveillance System (GLASS); Global Research and Development priority setting for AMR; Access Watch Reserve (AWaRe); and Global Antibiotic Research and Development Partnership (GARDP). 7 Murray et al. estimated that 4.95 million deaths occurred in the year 2019 as a result of antibiotic resistance. 6 Identied Klebsiella pneumoniae, as a common pathogen accounting for 29% of all reported bacterial infections. 7 Klebsiella pneumoniae has recently been identied as a bacteria of concern, as mentioned in several reports, studies, and reviews, and is further substantiated by the resistance map (Fig. 1) generated from the CDDEP (Centre for Disease Dynamics, Economics and Policy). 10 Unfortunately,

Virulent Enterobacterale strains expressing serine and metallo-β-lactamases (MBL) genes have emerged responsible for conferring resistance to hard-to-treat infectious diseases. One strategy that exists is to develop β-lactamase inhibitors to counter this resistance. Currently, serine β-lactamase inhibitors (SBLIs) are in therapeutic use. However, an urgent global need for clinical metallo-β-lactamase inhibitors (MBLIs) has become dire. To address this problem, this study evaluated BP2, a novel beta-lactam-derived β-lactamase inhibitor, co-administered with meropenem. According to the antimicrobial susceptibility results, BP2 potentiates the synergistic activity of meropenem to a minimum inhibitory concentration (MIC) of ≤1 mg/L. In addition, BP2 is bactericidal over 24 h and safe to administer at the selected concentrations. Enzyme inhibition kinetics showed that BP2 had an apparent inhibitory constant (Kiapp) of 35.3 µM and 30.9 µM against New Delhi Metallo-β-lactamase (NDM-1) and ...

Virulent Enterobacterale strains expressing serine and metallo-β-lactamases (MBL) genes have emerged responsible for conferring resistance to hard-to-treat infectious diseases. One strategy that exists is to develop β-lactamase inhibitors to counter this resistance. Currently, serine β-lactamase inhibitors (SBLIs) are in therapeutic use. However, an urgent global need for clinical metallo-β-lactamase inhibitors (MBLIs) has become dire. To address this problem, this study evaluated BP2, a novel beta-lactam-derived β-lactamase inhibitor, co-administered with meropenem. According to the antimicrobial susceptibility results, BP2 potentiates the synergistic activity of meropenem to a minimum inhibitory concentration (MIC) of ≤1 mg/L. In addition, BP2 is bactericidal over 24 h and safe to administer at the selected concentrations. Enzyme inhibition kinetics showed that BP2 had an apparent inhibitory constant (Kiapp) of 35.3 µM and 30.9 µM against New Delhi Metallo-β-lactamase (NDM-1) and ...

Virulent Enterobacterale strains expressing serine and metallo-β-lactamases (MBL) genes have emerged responsible for conferring resistance to hard-to-treat infectious diseases. One strategy that exists is to develop β-lactamase inhibitors to counter this resistance. Currently, serine β-lactamase inhibitors (SBLIs) are in therapeutic use. However, an urgent global need for clinical metallo-β-lactamase inhibitors (MBLIs) has become dire. To address this problem, this study evaluated BP2, a novel beta-lactam-derived β-lactamase inhibitor, co-administered with meropenem. According to the antimicrobial susceptibility results, BP2 potentiates the synergistic activity of meropenem to a minimum inhibitory concentration (MIC) of ≤1 mg/L. In addition, BP2 is bactericidal over 24 h and safe to administer at the selected concentrations. Enzyme inhibition kinetics showed that BP2 had an apparent inhibitory constant (Kiapp) of 35.3 µM and 30.9 µM against New Delhi Metallo-β-lactamase (NDM-1) and ...

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway...

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway...

Prediction of the stability of proteins is a critical step in the goal of protein design. The Partial Least Squares (PLS) regression method was used to predict the thermal stability of protein variants produced by single point mutations. The efficacy of this method was tested by using data on changes in free energy of unfolding (ddG) of mutations of phage T4 lysozyme. These data were obtained from the ProTherm database. PLS regression models were built by using physico-chemical descriptors. Structural information can also be incorporated into these models, if it is available. The models obtained in this manner were able to fit the experimental data on free energy of unfolding with an rms error of 0.42 kcal/mol and a two state classification accuracy of 89.9%. The magnitude of the rms error for these models is comparable to the magnitude of the expected errors in the experimental data. However, it was observed that less complex models, obtained from PLS models of lower rank, are more robust. The predictive rms error for these robust models were 1.2 kcal/mol (without structural information) and 1.1 kcal/mol (with structural information).

ABSTRACT: Prediction of the stability of proteins is a critical step in the goal of protein design. The Partial Least Squares (PLS) regression method was used to predict the thermal stability of protein variants produced by single point mutations. The efficacy of this method was tested by using data on changes in free energy of unfolding (ddG) of mutations of phage T4 lysozyme. These data were obtained from the ProTherm database. PLS regression models were built by using physico-chemical descriptors. Structural information can also be incorporated into these models, if it is available. The models obtained in this manner were able to fit the experimental data on free energy of unfolding with an rms error of 0.42 kcal/mol and a two state classification accuracy of 89.9%. The magnitude of the rms error for these models is comparable to the magnitude of the expected errors in the experimental data. However, it was observed that less complex models, obtained from PLS models of lower rank...

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins.

New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all major beta-lactams including carbapenems. Amino acid substitutions outside the active site reportedly affect NDM beta-lactamase activities. Here, the effect of amino acid substitutions in the possible omega-like loop region of NDM-5 has been elucidated. Overall, three substitution mutations near active site of NDM-5 were done, namely, E152A, S191A and D223A and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable host. Among the three mutants, E152A substitution on a loop near the active site resulted in significant reduction in beta-lactam antibiotic resistance as compared to NDM-5 that compelled us to conduct further studies on the E152A-substituted NDM-5. The purified NDM-5 was able to hydrolyse all the beta-lactams tested whereas the E152A mutation suppressed its activities. NDM-5 showed maximum k cat /K m ratio against penicillins and carbapenems and had lower K m as compared to NDM-5 E152A. Though, the amino acid substitution did not affect the overall folding pattern of NDM-5, significant differences in thermal stability between the wild-type and mutated protein were observed. Therefore, we infer that the E152 residue is important in regulating the beta-lactam hydrolysing properties of NDM-5.