Physical and molecular bases of protein thermal stability and cold adaptation

Pucci, Fabrizio; Rooman, Marianne

doi:10.1016/j.sbi.2016.12.007

Cited by 132 publications

(116 citation statements)

References 108 publications

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“…Even more difficult is the identification of the principles that confer protein structures elevated stabilities when exposed to unusual external conditions (e.g., very high/low temperature or chemical denaturants) . Although the development of proteins endowed with enhanced stabilities would have a tremendous impact in chemistry, biology, and medicine, their rational design is still an elusive goal .…”

Section: Introductionmentioning

confidence: 99%

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

et al. 2019

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The definition of the structural basis of protein thermostability represents a major topic in structural biology and protein chemistry. We have recently observed that proteins isolated from thermophilic organisms show a better adherence to the fundamental rules of protein topology previously unveiled by Baker and coworkers (Koga et al. Nature. 2012; 491: 222–227). Here, we explored the possibility that ad hoc modifications of a natural protein following these rules could represent an efficient tool to stabilize its structure. Hence, we here designed and characterized novel variants of Escherichia coli thioredoxin (EcTrx) using a repertoire of biophysical/structural techniques. Trx chimeric variants were prepared by replacing the loop of EcTrx with the corresponding ones present in the Trxs isolated from Sulfolobus solfataricus and Sulfolobus tokodaii that show a better adherence to the topological rules. Interestingly, although the loop sequences of these proteins did not display any significant similarity, their insertion in EcTrx induced a remarkable stabilization of the protein (≥10 °C). The crystallographic structure of one of these variants corroborates the hypothesis that the optimization of the loop size is the driving force of the observed stabilization. The remarkable stabilization of the two novel chimeric Trxs, generated by applying the topological rules, represents the proof of concept that these rules may be used to stabilize natural proteins through the ad hoc optimization of the loop size. Based on the present results, we propose a novel protocol of protein stabilization that can be potentially applied to other proteins.

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Section: Introductionmentioning

confidence: 99%

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

et al. 2019

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“…The sigmoid fitting of this data estimated its melting temperature (Tm) as 50.5 ± 0.02 °C. This value falls within the range of 20–60 °C for proteins from a mesophile such as Pseudomonas aeruginosa …”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of Homoserine Dehydrogenase from Pseudomonas aeruginosa

Kim

Nguyen

Yang

2019

Bulletin Korean Chem Soc

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Homoserine dehydrogenase (HSD) catalyzes the reduction of l‐aspatate‐4‐semaldehyde (l‐ASA) to l‐homoserine (l‐HSE) or oxidation reversely, which is a part of the aspartate pathway synthesizing threonine, isoleucine, and methionine in vivo. HSD has gained much interest in medical application since HSD is a well‐established target for pesticides and antibiotics. In addition, HSD is also valuable in industrial application for l‐lysine production. In this study, HSD from Pseudomonas aeruginosa (PaHSD) was overexpressed in Escherichia coli, and purified to homogeneity for molecular and biochemical characterization. PaHSD exhibits the comparable activity of l‐HSE oxidation for both types of cofactors, NAD+ and NADP+, but at the same time shows interesting differences. The kcat/Km value of NADP+ was ~1.8 times larger than that of NAD+, while the kcat/Km value of l‐HSE was ~1.4 times larger with NAD+ than with NADP+. Notably, the Km value is about three times smaller for NADP+, while Vmax is about 1.7 times larger for NAD+, which implies that while NADP+ binds more strongly to the active site, NAD+ has a faster turnover. Size‐exclusion chromatography analysis showed PaHSD forms tetramers, as opposed to HSDs from other species forming dimers. In the circular dichroism analysis, PaHSD showed a typical spectrum of α/β structure, and its melting temperature was determined to be 50.5 °C from the thermal denaturation test at 222 nm. The molecular and biochemical features of PaHSD revealed in this study provide the basis for the future study of amino acid metabolism, and its application to industrial and medical purposes.

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“…Besides, Marqusee and co-workers found that high kinetic stability is related to unusual resistance to proteolysis; however, they do not find any common structural features that account for proteolytic resistance (Park et al 2007). Several works have pointed out the importance of ionic, hydrophobic, π-π, and cation-π interactions in stabilizing proteins in the hightemperature regime, and conclude that the involved mechanisms cannot be fully rationalized on the basis of the sum of individual contributions but require a global analysis in terms of interaction networks (Karshikoff et al 2015;Pucci and Rooman 2017).…”

Section: Kinetic Stability: the Two-state Irreversible Modelmentioning

confidence: 99%

Kinetic stability of membrane proteins

2017

Biophys Rev

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Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

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Physical and molecular bases of protein thermal stability and cold adaptation

Cited by 132 publications

References 108 publications

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

Biochemical Characterization of Homoserine Dehydrogenase from Pseudomonas aeruginosa

Kinetic stability of membrane proteins

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