The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient

Abstract

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of proteinprotein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.

Efeito da composição do solvente sobre a estabilidade de proteínas em soluções aquosas

“…Como o estado D tem maior exposição de sítios para ligação de uréia que o estado N 47 , a hidratação da proteína mediada pela uréia deve ser maior no estado D que no estado N, de tal forma que o estado D deve ser favorecido pela uréia. O hidrocloreto de guanidina também forma ligações de hidrogênio com grupos das cadeias laterais dos aminoácidos e das ligações peptídicas 48,49 , que também estão mais acessíveis ao caotrópico no estado desnaturado.…”

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

“…When the negative B 22 value further decreases, solution conditions are obtained enhancing particle-particle attractions, and as a result in a number of cases amorphous protein precipitation is observed [51][52][53].…”

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

“…L-Arg is known to be able to prevent aggregation caused by hydrophobic interactions (Tsumoto et al 2004), by masking hydrophobic regions and reducing attractive intermolecular interactions (Ho et al 2003). In silico modelling has shown that the guanidium group of L-Arg interacts with hydrophobic patches of protein while the polar carboxyl part of L-Arg forms a hydrophilic layer (Li et al 2010).…”

“…Tag removal from IT1 VP1, containing a hydrophobic epitope, results in only free GST and aggregated IT1 VP1. acids (Ho et al 2003;Leibly et al 2012;Tsumoto et al 2004), osmolytes (Leibly et al 2012), and molecular chaperones (Lanckriet and Middelberg 2004;Prashar et al 2011;Serno et al 2011) as detailed in Table 3.2. Thrombin activity in the different buffer conditions was assessed and confirmed that complete digestion of the GST-VP1 fusion was achieved between pH 7.0 and pH 9.0, irrespective of the additive present.…”