A Proteomic Approach toward the Selection of Proteins with Enhanced Intrinsic Conformational Stability

Abstract

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.

Conformational States and Protein Stability from a Proteomic Perspective

“…On the other hand, a less discussed aspect relates to . See (Prosinecki et al, 2006) for further details.…”

“…This hypothesis has been originally put forward following the profiling of Sulfurisphaera protein 'stabilome': the identified proteins participate in cellular processes (e.g.. defence against reactive oxygen species, nucleic acid protection and energy production) in which some key proteins have enhanced thermal stabilities, which may relate to their importance on the metabolism of the organism. For example, finding proteins involved in cellular detoxification processes to be hyperstable, such as iron superoxide dismutase and peroxiredoxin, may be rationalized in terms of the fact that proteins dealing with reactive oxygen species need enhanced conformational stabilities, namely to withstand and minimize possible oxidative modifications which could lead to protein inactivation (Prosinecki et al, 2006). Interestingly, oxidative stress related proteins such as Fe and Mn superoxide dismutase, catalase and glutathione reductase were also identified as survivors in proteolysis resistance studies in E. coli proteome (Park et al, 2007).…”

“…Recently, Gomes and co-workers, have established a novel approach aimed at profiling a proteome for its most intrinsically stable proteins (Prosinecki et al, 2006). The methodology was implemented on the proteome of a hyperthermophile, as the high optimal growth temperatures of these organisms (>80°C) make them valuable sources of proteins with intrinsically high stability.…”

“…The increase in the specific activity shows that upon 96 h perturbation the proteins retain their biological function and presumably their native fold. See (Prosinecki et al, 2006) for further details.…”

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from Thermococcus onnurineus NA1

“…In our previous study, we applied shotgun proteomic to screen hyperthermostable proteins of T. onnurineus NA1 and identified 150 hyperthermostable candidate proteins including fructose-1,6-bisphosphatase (TON_1497). Hyperthermostable protein sets were enriched and fractionated by a heat-treatment and centrifugation method (Prosinecki et al, 2006).…”