DIFFERENTIAL STABILIZING EFFECTS OF BUFFERS ON STRUCTURAL STABILITY OF BOVINE SERUM ALBUMIN AGAINST UREA DENATURATION

The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal. Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As found in MOPS and HEPES buffers, the highest stability of BSA can be attributed to the presence of morpholine and piperazine rings, respectively, in their structures. These groups might have produced a hydrophobic environment around the protein surface, thus stabilizing protein conformation against urea denaturation.

Extended ɛ34 Phage TSP Renatures After Urea-acid Unfolding

In antimicrobial-peptide/protein engineering, understanding the peptide/protein’s adaptability to harsh environmental conditions such as urea, proteases, fluctuating temperatures, high salts provide enormous insight into the pharmacokinetics and pharmacodynamics of the engineered peptide/protein and its ability to survive the harsh internal environment of the human body such as the gut or the harsh external environment to which they are applied. A previous work in our laboratory demonstrated that our cloned Eɛ34 TSP showed potent antimicrobial activity against Salmonella newington, and more so, could prevent biofilm formation on decellularized tissue. In this work, the effects of urea-acid on the Eɛ34 stability is studied, and the results demonstrates that at lower pHs of 3 and 4 with urea the protein was denatured into monomeric species. However, the protein withstood urea denaturation above pH of 5 and thus remained as trimeric protein. The mechanism of denaturation of Eɛ34 TSP seems to show that urea denatures proteins by depleting hydrophobic core of the protein by directly binding to the amide units via hydrogen bonds. The results of our in-silico investigation determined that urea binds with Eɛ34 TSP with relative free energies range of -3.4 to -2.9 kcal/mol at the putative globular head binding domain of the protein. The urea molecules interacts with with the protein’s predicted hydrophobic core, thus, disrupting and exposing the shielded hydrophobic moieties of Eɛ34 TSP to the solvent. We further showed that after the unfolding of Eɛ34 TSP via urea-acid, renaturation of the protein to its native conformation was possible within few hours. This unique characteristic of refolding of Eɛ34 TSP which is similar to that of the P22 phage tailspike protein is of special interest to protein scientists and can also be exploited in antimicrobial-protein engineering.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

TMAO: Protecting Proteins from Feeling the Heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but the effect of TMAO against high temperature has not been addressed. To delineate the effect of TMAO on folded and unfolded ensembles at different temperatures, we study a mutant of the well-characterized, fastfolding model protein B (PRB). We have carried out extensive, >190 µs in total, all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations captured folding and unfolding events and show an increased stability of PRB in presence of TMAO. At higher TMAO concentration, intermediate ensembles are gradually more favored over the unfolded state. Quantifying TMAO-water interactions revealed that at a low concentration threshold, TMAO forms a shell near but not at the protein surface, disrupting the water network and increasing hydration of the protein to help stabilize it. Intriguingly, we found that there are intermittent interactions between TMAO and certain protein side chains with preferred TMAO orientations. Although previous studies have proposed such interactions, the long time scales we study here help to highlight the protein’s sensitivity to local environment, particularly hydration, and raise questions about how even transient interactions could couple protein stability to TMAO effects.

TMAO: Protecting Proteins from Feeling the Heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but the effect of TMAO against high temperature has not been addressed. To delineate the effect of TMAO on folded and unfolded ensembles at different temperatures, we study a mutant of the well-characterized, fastfolding model protein B (PRB). We have carried out extensive, >190 µs in total, all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations captured folding and unfolding events and show an increased stability of PRB in presence of TMAO. At higher TMAO concentration, intermediate ensembles are gradually more favored over the unfolded state. Quantifying TMAO-water interactions revealed that at a low concentration threshold, TMAO forms a shell near but not at the protein surface, disrupting the water network and increasing hydration of the protein to help stabilize it. Intriguingly, we found that there are intermittent interactions between TMAO and certain protein side chains with preferred TMAO orientations. Although previous studies have proposed such interactions, the long time scales we study here help to highlight the protein’s sensitivity to local environment, particularly hydration, and raise questions about how even transient interactions could couple protein stability to TMAO effects.