Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions

Monera, Oscar D.; Kay, Cyril M.; Hodges, Robert S.

doi:10.1002/pro.5560031110

Cited by 350 publications

(281 citation statements)

References 32 publications

Supporting

Mentioning

267

Contrasting

Order By: Relevance

“…In these cases, the pH dependence of homodimer and heterodimer formation suggests that charge-charge interactions play a role in determining specificity. The salt dependence of leucine zipper stability also indicates that ionic effects are involved in stabilizing and/or destabilizing leucine zippers (Monera et al, 1994;Zhou et al, 1994b;Kohn et al, 1995;Yu et al, 1996). However, our results indicate that other factors involving the e and g positions can also play a significant role in determining dimerization specificity.…”

Section: Each Row Shows Results For One Cl' Fusion Protein Expressementioning

confidence: 61%

“…Indeed, a linear relationship between stability and the number of attractive and repulsive ionic interactions has been shown in vitro for a set of model coiled coils (Monera et al, 1994). The patterns of which mutants can titrate each other's activities should provide an additional test of the idea that charge complementarity is sufficient to predict dimerization specificity.…”

Section: Each Row Shows Results For One Cl' Fusion Protein Expressementioning

confidence: 99%

“…The properties of many leucine zipper mutants appear to support this idea (Schuermann et al, 1991;John et al, 1994;Zhou et al, 1994aZhou et al, , 1994b. Indeed, the stability of a set of two-stranded coiled-coil peptides was related linearly to the number of attractive and repulsive interchain and intrachain interactions (Monera et al, 1994). However, the contributions of ionic interactions between charged end groups remains controversial .…”

mentioning

confidence: 99%

“…NMR measurements of pK, shifts of glutamates involved in ion pairs in the GCN4 structure suggest that the net contributions of interchain Glu-Lys electrostatic interactions to coiled-coil stability are either insignificant or slightly destabilizing (Lumb & Kim, 1995). The relative and absolute contributions of attractive and repulsiveg-e' ionic interactions are also clearly dependent on solution conditions (Monera et al, 1994;Yu et al, 1996). Models for how different amino acid side chains at the e and g positions determine dimerization specificity should be able to predict the formation of homodimers and heterodimers among a set of related sequences that vary only at these positions.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

View full text Add to dashboard Cite

Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of >65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing A repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (>dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type I11 mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.Keywords: dimerization specificity; leucine zippers; protein structure; recombinant fusion proteins; site-directed mutagenesis a-Helical coiled coils are involved in the assembly of a wide variety of proteins. A subclass of the coiled-coils known as leucine zippers are found as short dimerization motifs in many eukaryotic transcription factors. Leucine zipper sequences are characterized by leucine appearing in every seventh position (4 over four to five heptad repeats (abcdefg), (for a review, see Hurst, 1994). Leucine zippers fold into dimeric, parallel coiled-coils, where each heptad forms two a-helical turns. Because of their small size and simple structure, leucine zippers and other short a-helical coiled coils have been used extensively as a model system to study how amino acid sequences specify structure (e.g., see Hodges et al., 1988;

show abstract

Section: Each Row Shows Results For One Cl' Fusion Protein Expressementioning

confidence: 61%

Section: Each Row Shows Results For One Cl' Fusion Protein Expressementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

View full text Add to dashboard Cite

show abstract

“…Among this series, the guanidinium ion (Gdm + ) is the most widely used chemical agent in protein denaturation due to its strong destabilizing effect and high solubility in water. Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22).…”

mentioning

confidence: 99%

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Ding

Mukherjee

Chen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still remains challenging because it requires assessment of all relevant interactions, such as ion-protein, ion-water, and ion-ion interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can promote α-helix formation in certain peptides.T he stability of a protein, or more precisely, the free energy difference between its folded and unfolded states, can be modulated by various solution properties. For example, addition of another solute to a protein solution can result in either a decrease or an increase in this protein's stability (1-3). The most noticeable example in this regard is the Hofmeister series (4-6), a group of ions that are ranked based on their protein-denaturing abilities. Among this series, the guanidinium ion (Gdm + ) is the most widely used chemical agent in protein denaturation due to its strong destabilizing effect and high solubility in water. Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22). In particular, it has been hypothesized that Gdm + , which exists in aqueous solution as a rigid, flat object (12,18,19), can engage in stacking interactions with amino acids consisting of planar side chains, such as arginine (Arg) (19), asparagine (Asn) (11,20), glutamine (Gln) (11,20), and aromatic residues (20)(21)(22). However, to the best of our knowledge, the only experimental evidence that supports this hypothesis comes from crystallographic data (20)(21)(22), which show that the guanidinium group of Arg is more frequently found to be stacked against the side chain of tyrosine (Tyr) or tryptophan (Trp) in proteins. Therefore, additional experimental studies that can directly probe such stack interactions in solution are needed.Another ion in the Hofmeister series that has a similar proteindenaturing capability to Gdm + is tetrapropylammonium (TPA + ) (23). However, a recent study by Dempsey et al. (24) found that although TPA + , like Gdm + , can denature a tryptophan (Trp) zipper β-hairpin (i.e....

show abstract

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: Role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

et al. 1999

View full text Add to dashboard Cite

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal dena-turation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5°C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular di-chroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 Å crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures. Proteins 1999;34:356-368.

show abstract

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions

Cited by 350 publications

References 32 publications

Oligomerization properties of GCN4 leucine zipper e and g position mutants

Oligomerization properties of GCN4 leucine zipper e and g position mutants

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: Role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

Contact Info

Product

Resources

About