Comparative thermal unfolding study of psychrophilic and mesophilic subtilisin-like serine proteases by molecular dynamics simulations

Du, Xing; Sang, Peng; Yuan, Xia; Li, Yang; Liang, Jing; Ai, Shi Meng; Ji, Xing; Fu, Yunxin; Liu, Shu Qun

doi:10.1080/07391102.2016.1188155

Cited by 19 publications

(16 citation statements)

References 88 publications

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“…13). These observations fit our ideas concerning the unfolding pathway and the role of the Ca1 binding site 25 , as a compromised Ca1 binding site could lead to destabilisation of a possible unfolding initiation point at helix D 29 . In addition, low pH values could destabilise further interactions important for the stability of the protein structure but less so for the intermediate state.…”

Section: Discussionsupporting

confidence: 87%

“…Combined with the high activation energy of the second transition, this could indicate that the intermediate still retains the calcium-binding site 3. Even though VPR ΔC unfolds in a rather cooperative manner, an elucidation of the chronological order of events during the thermal unfolding of wild type VPR using MD has been reported 29 . There, helix D close to the Ca1 binding site appeared to be the initiation point of thermal unfolding.…”

Section: Stabilitymentioning

confidence: 99%

“…Thus, placing the initiation point of thermal unfolding around that mutation. MD-simulation at different temperatures have suggested that the initiation point of unfolding is helix D 29 . Helix D is proximal to the Ca1 site, a site that is important for the stability of the structure 25,30,31 .…”

Section: Stabilitymentioning

confidence: 99%

“…Why this mutation had destabilizing effects when added to the wild type but stabilizes the structure when VPR ΔC _N3P/I5P is the template, is an interesting observation. The site of the N238P mutation is on a loop at the C-end of helix E, a helix that may well be one of the more stable parts of the structure 29 . The mutation does cause loss of H-bond potentials that could be the reason for the detrimental effects observed on the wild type template.…”

Section: Stabilitymentioning

confidence: 99%

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Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Óskarsson

Sævarsson

Kristjánsson

2020

Sci Rep

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protein stability is a widely studied topic, there are still aspects however that need addressing. in this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VpR, using the thermostable structural homologue AQUi as a template. four locations for proline substitutions were chosen to imitate the structure of AQUi. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPR Δc _N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. in addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.

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Section: Discussionsupporting

confidence: 87%

Section: Stabilitymentioning

confidence: 99%

Section: Stabilitymentioning

confidence: 99%

Section: Stabilitymentioning

confidence: 99%

See 2 more Smart Citations

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Óskarsson

Sævarsson

Kristjánsson

2020

Sci Rep

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“…In summary, our combined use of in vitro methods (kinetic experiments and thermal stability analyses) and in silico MDS analysis has provided further insights into the mechanisms by which orthologous proteins become adapted for function in widely different thermal conditions. Most prior studies that used MDS methods to examine temperature effects on proteins compared distantly related species (7,(29)(30)(31)(32); our work, with 12 differently adapted congeners belonging to five molluscan genera, allowed a multilineage analysis of convergent adaptation in flexibility. We show that protein flexibility as indexed by ΔRMSD and ΔRMSF is a common outcome of adaptive changes in amino acid sequence and that regions involved in large conformational changes during binding and catalysis are especially important features of the adaptive response.…”

Section: And Summarized Inmentioning

confidence: 99%

Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs

Dong

Liao

Meng

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

237

143

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Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

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Ordered‐domain unfolding of thermophilic isolated β subunit ATP synthase

López‐Pérez,

de Gómez‐Puyou,

Nuñez

et al. 2023

Protein Science

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The flexibility of the ATP synthase's β subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated β subunit (Tβ) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tβ shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the β-sheet residual structure at high temperature. We determined that part of the stability origin of Tβ is due to a transversal hydrophobic array that crosses the β-barrel formed at the N-terminal domain and the Rossman fold of the nucleotidebinding domain (NBD), while the helix bundle of the C-terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase.

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Comparative thermal unfolding study of psychrophilic and mesophilic subtilisin-like serine proteases by molecular dynamics simulations

Cited by 19 publications

References 88 publications

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs

Ordered‐domain unfolding of thermophilic isolated β subunit ATP synthase

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