Chemically crosslinked protein dimers: Stability and denaturation effects

Abstract

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; I ,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethy1)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCI) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, e-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.

Cisplatin Affects the Conformation of Apo Form, not Holo Form, of BRCA1 RING Finger Domain and Confers Thermal Stability

“…74 -838), it was consistent with the previous study, showing that the Zn 2 þ finger domain formed a thermostable structure [53] [60]. Furthermore, the increased thermostability of cisplatin -BRCA1 adducts by 138 probably resulted from the thermodynamically stabilizing contribution of intermolecular cross-links [61].…”

Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase

“…As monitored by both CD and fluorescence, the N84Q denaturation curves are not only shifted in terms of [GdnHCl] 1/2 , but also slightly steeper in slope (m). A study by Byrne and Stites (23) proposes that a change in the slope of a denaturation curve may be related to the amount of hydrophobic area exposed during the course of denaturation of a protein. A steeper slope indicates that a protein exposed more hydrophobic area during denaturation, implying that it started with less of that hydrophobic area exposed.…”

“…The T m of N84Q LCAT has shifted down by about 3.5°C. This difference in T m corresponds to a difference of around 0.5 kcal/mol of free energy change in calorimetry studies of other proteins (23). It is important to note that the N84Q mutant does have some activity at this point, although the same mutant has no activity after the protein purification procedure, as shown by the enzymatic activity assays.…”

Bridged Analogues for p53-Dependent Cancer Therapy Obtained by S-Alkylation

“…These include hydrazone bridge (Cabezas and Satterthwait, 1999), oxime bridge (Haney et al, 2011), 1,4-disubstituted-[1,2,3]-triazole linkage (Holland-Nell and Meldal, 2011; Ingale and Dawson, 2011; Kawamoto et al, 2012; Lau et al, 2014c; Lau et al, 2014b; Lau et al, 2014a; Lau et al, 2015b; Scrima et al, 2010), metal chelation (Ghadiri and Choi, 1990; Ruan et al, 1990), disulfide bond formation (Almeida et al, 2012; Jackson et al, 1991; Leduc et al, 2003), lactam ring formation (Fujimoto et al, 2008; Geistlinger and Guy, 2001; Geistlinger and Guy, 2003; Houston, Jr. et al, 1995; Osapay and Taylor, 1992; Phelan et al, 1997) and S-alkylation based staples employing either α-haloacetamide alkylation of single cysteine (Brunel and Dawson, 2005; Cardoso et al, 2007; Galande et al, 2004; Woolley, 2005) or bridging two cysteines with bis-S-alkylating linker(s) (de Araujo et al, 2014; Jo et al, 2012; Muppidi et al, 2011b; Muppidi et al, 2011a; Muppidi et al, 2012; Spokoyny et al, 2013; Szewczuk et al, 1992; Timmerman et al, 2005; Wilkinson et al, 2007; Zhang et al, 2007; Zhang et al, 2008). Among these, the last seems to be most flexible approach as a wide range of inexpensive bis-thiol-reactive linkers is commercially available, including rigid aromatic derivatives (Chua et al, 2015; Jo et al, 2012; Muppidi et al, 2011b; Muppidi et al, 2011a; Muppidi et al, 2012; Timmerman et al, 2005; Zhang et al, 2007) and aliphatic counterparts (Byrne and Stites, 1995; Chua et al, 2015; Lindman et al, 2001; Wilkinson et al, 2007). In addition, the availability of various cysteine homologs: (L)Cys, (D)Cys, (L)homoCys, (D)homoCys, (L)Pen, and (D)Pen provides an additional option for “fine tuning” of pre-selected active derivatives.…”