Conformational Stability and Denaturation Processes of Proteins Investigated by Electrophoresis under Extreme Conditions

Abstract

The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.

Potensi dan Karakteristik Bubuk Cangkang Telur yang Dibuat dengan Perendaman Asam Alami

“…Penggunaan pretreatment asam menghasilkan nilai protein yang lebih rendah daripada kontrol, hal ini karena senyawa asam diduga mampu menyebabkan denaturasi pada protein Bubuk cangkang telur selama proses perendaman. Denaturasi protein bisa disebabkan oleh banyak faktor, antara lain kondisi fisikokimia, pH, kekuatan ion, konstanta dielektrik, radiasi dan agen kimia yang mengikat dengan ikatan intramolekular non kovalen sebagai contoh adalah larutan senyawa organik (Masson dan Lushchekina, 2022). Penggunaan larutan asam pada penelitian termasuk asam lemah sehingga belum sepenuhnya mampu memutus ikatan protein yang ada, sehisngga proses deproteinasi bubuk cangkang telur belum maksimal (Arianto et al, 2022)…”

Osmolyte induced protein stabilization: modulation of associated water dynamics might be a key factor

“…Alternatively, it may assume an intermediate conformation or aggregate into accumulating b-amyloid fibrils, which have been observed to exhibit toxicity towards cells. 1 Nevertheless, Nature is well-equipped with many defense mechanisms to combat the deleterious effect of these perturbations, and osmolytes play an important role in this process. The osmolytes are small natural organic molecules, which are accumulated in the cells while in stress to protect proteins and enzymes.…”

“…The measured decay time constants (t i ) and the corresponding amplitudes (a i ) of fluorescence transients across the emission spectrum were used along with the steady state emission, F, to construct the time-resolved emission spectra (TRES) according to eqn (1) with its fitting to a log-normal function, where I 0 (l,t) represents the time-dependent intensities at various wavelengths (l).…”

Conformational Stability of Peroxidase from latex ofArtocarpus lakoocha: Influence of pH, Chaotropes, and Temperature

“…Folding and conformational stability of proteins are primarily based on their amino acid sequence as well as cellular external variables such as temperature, pH, and chemical denaturants (Anfinsen, 1973;Masson & Lushchekina, 2022;Mohamed, El-Badry, Drees, & Fahmy, 2008).…”

“…Folding and conformational stability of proteins are primarily based on their amino acid sequence as well as cellular external variables such as temperature, pH, and chemical denaturants (Anfinsen, 1973; Masson & Lushchekina, 2022; Mohamed, El-Badry, Drees, & Fahmy, 2008). To predict the behavior of protein folding, it is important to understand the effect of these external variables on the thermodynamics of the folding process.…”