Ion Pairs Significantly Stabilize Coiled-coils in the Absence of Electrolyte

Yu, Yinan; Monera, Oscar D.; Hodges, Robert S.; Privalov, Peter L.

doi:10.1006/jmbi.1996.0030

Cited by 70 publications

(69 citation statements)

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“…The increased protonation (neutralization) of the acidic residues at low pH, as well as screening of electrostatic interactions at higher salt concentrations, is expected to eliminate or reduce the effects of the repulsive Asp70 -Glu9 interaction, in agreement with the experimental observations. Of course, protonation of the acidic residues would also result in a reduction of the stabilizing electrostatic interactions (i.e., salt bridges) [43,44]. Consequently, the overall stability of Stx2 B pentamer would be expected to decrease with decreasing pH, which is consistent with our experimental observations.…”

Section: ϩ9supporting

confidence: 88%

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

Kitova

Daneshfar

Marcato

et al. 2005

J. Am. Soc. Mass Spectrom.

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The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B 5 homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60°C and subunit concentrations ranging from 5 to 85 M, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 M, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B 5 ions of Stx2 at the ϩ12 and ϩ13 charge states proceeds, at reaction temperatures of 120 to 180°C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B 5 ions at the ϩ12 and ϩ13 charge states were compared with values reported for the corresponding B 5 ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E a ) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E a values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins. (J Am Soc Mass Spectrom 2005, 16, 1957

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Section: ϩ9supporting

confidence: 88%

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

Kitova

Daneshfar

Marcato

et al. 2005

J. Am. Soc. Mass Spectrom.

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“…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%

“…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.…”

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confidence: 99%

Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

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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;

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“…[19][20][21] It is likely that charge repulsion plays a role in destabilizing the ecE and ecK homotetramers since, in both cases, the peptides are largely unfolded (see Fig. 2) under conditions in which the parent sequence is quite stable.…”

Section: Resultsmentioning

confidence: 99%

Design of a heterotetrameric coiled coil

et al. 2009

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We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407.We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.

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Ion Pairs Significantly Stabilize Coiled-coils in the Absence of Electrolyte

Cited by 70 publications

References 0 publications

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

Stability of the homopentameric b subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry

Oligomerization properties of GCN4 leucine zipper e and g position mutants

Design of a heterotetrameric coiled coil

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