A conformationally isoformic thermophilic protein with high kinetic unfolding barriers

Mishra, Rajesh; Olofsson, Linus; Karlsson, Martin; Carlsson, Uno; Nicholls, Ian A.; Hammarström, Per

doi:10.1007/s00018-008-7517-4

Cited by 7 publications

(7 citation statements)

References 39 publications

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“…This leads to a slightly lower stability value for GdmCl compared to that for urea, despite the higher concentration of surfactant. Use of a stronger denaturant typically leads to higher extrapolated values of unfolding rates, − though the reasons for this are not clear.…”

Section: Resultsmentioning

confidence: 99%

A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways

2012

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The outer membrane protein OmpA from Escherichia coli can fold into lipid vesicles and surfactant micelles from the urea-denatured state. However, a complete kinetic description of the folding and unfolding of OmpA, which can provide the basis for subsequent protein engineering studies of the protein's folding pathway, is lacking. Here we use two different denaturants to probe the unfolding mechanism of OmpA in the presence of the surfactant octyl maltoside (OM). Unfolding of OmpA in the presence of micelles, achieved with the potent denaturant guanidinium chloride (GdmCl), leads to single-phase unfolding. In contrast, OmpA unfolds in urea only below OM's critical micelle concentration, and this occurs in different phases, which we attribute to the existence of states that have bound different amounts of surfactant, from completely "naked" to partly covered by surfactant. Multiple parallel refolding phases are attributed to different levels of collapse prior to folding. Kinetic results used to derive the stability of OmpA in surfactant, using either urea or GdmCl as the denaturing agent, give comparable results and indicate a minimalist three-state folding scheme involving denatured state D, folding intermediate I, and native state N. N and I are stabilized by 15.6 and 2.6 kcal/mol, respectively, relative to D. The periplasmic domain of OmpA does not contribute to stability in surfactant micelles. However, BBP, a minimalist transmembrane β-barrel version of OmpA with shortened loops, is destabilized by ~10 kcal/mol compared to OmpA, highlighting loop contributions to OmpA stability.

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Section: Resultsmentioning

confidence: 99%

A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways

2012

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“…Structural flexibility of proteins from psychrophilic organisms result from low hydrophobicity in the protein core, low proline, and high glycine content (Hough and Dan-son1999;Feller 2003;Pica and Graziano 2016;Pucci and Rooman 2017). Thermophilic proteins, like Thermoanaerobacter brockii alcohol dehydrogenase (TBADH), show the involvement of additional salt bridges, hydrogen bonding, hydrophobicity, oligomerization, and strategically placed prolines to provide higher conformational stability than the mesophilic counterpart, Clostridium beijerinckii alcohol dehydrogenase (CBADH) (Bogin et al 1998;Mishra et al 2008). Similarly, acidophilic and alkaliphilic proteins show the preferences of acidic and basic amino acid residues respectively as their adaptive mechanism (Hough and Danson 1999;Reed et al 2013).…”

Section: Factors Responsible For Protein Stability In Extreme Environ...mentioning

confidence: 99%

Structural and functional adaptation in extremophilic microbial α-amylases

2022

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Maintaining stable native conformation of a protein under a given ecological condition is the prerequisite for survival of organisms. Extremophilic bacteria and archaea have evolved to adapt under extreme conditions of temperature, pH, salt, and pressure. Molecular adaptations of proteins under these conditions are essential for their survival. These organisms have the capability to maintain stable, native conformations of proteins under extreme conditions. The enzymes produced by the extremophiles are also known as extremozyme, which are used in several industries. Stability and functionality of extremozymes under varying temperature, pH, and solvent conditions are the most desirable requirement of industry. α-Amylase is one of the most important enzymes used in food, pharmaceutical, textile, and detergent industries. This enzyme is produced by diverse microorganisms including various extremophiles. Therefore, understanding its stability is important from fundamental as well as an applied point of view. Each class of extremophiles has a distinctive set of dominant non-covalent interactions which are important for their stability. Static information obtained by comparative analysis of amino acid sequence and atomic resolution structure provides information on the prevalence of particular amino acids or a group of non-covalent interactions. Protein folding studies give the information about thermodynamic and kinetic stability in order to understand dynamic aspect of molecular adaptations. In this review, we have summarized information on amino acid sequence, structure, stability, and adaptability of α-amylases from different classes of extremophiles.

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“…Observing the intermediate structures contributes to our understanding of protein folding and unfolding. − Biophysical techniques such as circular dichroism (CD), fluorescence spectroscopy, small-angle X-ray scattering, and nuclear magnetic resonance (NMR) spectroscopy allow us to monitor intermediate structures during conformational changes induced by thermal or chemical denaturation. − Unfortunately, protein folding and unfolding are generally fast, and most intermediate structures are temporary and difficult to detect. Recent research in the field has indicated that some proteins from hyperthermophiles are stabilized by their remarkably slow unfolding rate. − Therefore, we are able to detect the intermediates of these proteins because of their slow unfolding.…”

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confidence: 99%

“…Many hyperthermophilic proteins have considerable kinetic stability as measured by their high activation energy and slow unfolding kinetics. − Well-studied examples are pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf-PCP) and ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2). The unfolding rate of Pf-PCP is 10 7 times slower than that of mesophilic PCP from Bacillus amyloliquefaciens .…”

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confidence: 99%

Slow Unfolding Pathway of Hyperthermophilic Tk-RNase H2 Examined by Pulse Proteolysis Using the Stable Protease Tk-Subtilisin

et al. 2012

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The unfolding speed of some hyperthermophilic proteins is significantly slower than those of their mesostable homologues. Ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate. In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a superstable subtilisin-like serine protease from T. kodakarensis (Tk-subtilisin). Tk-subtilisin has enzymatic activity in highly concentrated guanidine hydrochloride (GdnHCl), in which Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 was completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) was degraded by Tk-subtilisin. Degradation products of Tk-RNase H2 created from pulse proteolysis during its unfolding were detected by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and mass spectrometry and constructed mimics of the unfolding intermediate of Tk-RNase H2 by protein engineering. The mimics were biophysically characterized. We found that the native state of Tk-RNase H2 (N-state) changed to the I(A)-state that was digested by Tk-subtilisin in the early stage of unfolding. In the slow unfolding pathway, the I(A)-state shifted to two intermediate forms, I(B)-state and I(C)-state. The I(B)-state was digested by Tk-subtilisin in the C-terminal region, but the I(C)-state was a Tk-subtilisin resistant form. These states gradually unfolded through the I(D)-state, in which the N-terminal region was digested. The results indicate that pulse proteolysis, by a superstable protease, was a suitable strategy and an effective tool for analyzing intermediate structures of proteins with slow unfolding properties. We also showed that the N-terminal region contributes to the slow unfolding of Tk-RNase H2, and the C-terminal region is important for folding and stability.

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A conformationally isoformic thermophilic protein with high kinetic unfolding barriers

Cited by 7 publications

References 39 publications

A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways

A Kinetic Analysis of the Folding and Unfolding of OmpA in Urea and Guanidinium Chloride: Single and Parallel Pathways

Structural and functional adaptation in extremophilic microbial α-amylases

Slow Unfolding Pathway of Hyperthermophilic Tk-RNase H2 Examined by Pulse Proteolysis Using the Stable Protease Tk-Subtilisin

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