A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase

Vaughan, Cara K.; Harryson, Pia; Buckle, Ashley M.; Fersht, Alan R.

doi:10.1107/s0907444902001567

Cited by 45 publications

(59 citation statements)

References 53 publications

(57 reference statements)

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“…Similar pK A shifts are found for salt bridges in proteins (Oliveberg et al, 1995;Vaughan et al, 2002) and for the His of the FYVE domain upon binding to the negatively charged lipid phosphatidyl(3)inositol . Determination of precisely how many H + are exchanged in the Lti30 binding process is yet precluded by our approximate estimate K d obs ðpHÞ: Even so, these data provide direct evidence that the interaction between Lti30 and membranes is indeed modulated by the ionization states of the flanking His residues.…”

Section: The Ph Dependence Of Vesicle Aggregation Corroborates the Insupporting

confidence: 60%

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

et al. 2011

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Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30's proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest.

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Section: The Ph Dependence Of Vesicle Aggregation Corroborates the Insupporting

confidence: 60%

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

et al. 2011

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“…2). This is lower than values previously seen for a buried salt bridge (6,14), but it does suggest a specific interaction indicating these residues are physically close (15).…”

Section: -Ht 3 Receptor Characterizationcontrasting

confidence: 47%

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Price

Millen

Lummis

2007

Journal of Biological Chemistry

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5-Hydroxytryptamine (5-HT) 3 and ␥-aminobutyric acid, type C (GABA C ) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which also includes nicotinic acetylcholine, GABA A , and glycine receptors. The details of how agonist binding to these receptors results in channel opening is not fully understood but is known to involve charged residues at the extracellular/transmembrane interface. Here we have examined the roles of such residues in 5-HT 3 and GABA C receptors. Charge reversal experiments combined with data from activation by the partial agonist ␤-alanine show that in GABA C receptors there is a salt bridge between Glu-92 (in loop 2) and Arg-258 (in the pre-M1 region), which is involved in receptor gating. The equivalent residues in the 5-HT 3 receptor are important for receptor expression, but charge reversal experiments do not restore function, indicating that there is not a salt bridge here. There is, however, an interaction between Glu-215 (loop 9) and Arg-246 (pre-M1) in the 5-HT 3 receptor, although the coupling energy determined from mutant cycle analysis is lower than might be expected for a salt bridge. Overall the data show that charged residues at the extracellular/transmembrane domain interfaces in 5-HT 3 and GABA C receptors are important and that specific, but not equivalent, molecular interactions between them are involved in the gating process. Thus, we propose that the molecular details of interactions in the transduction pathway between the binding site and the pore can differ between different Cys-loop receptors.There have been many biochemical and functional studies on different members of the Cys-loop family of ligand-gated ion channels, but the prototypic member of this family, the neuromuscular nicotinic acetylcholine (nACh) 2 receptor, is still the best understood. This is the only receptor for which reasonably high resolution structural information (4 Å) is available from cryo-electron microscopy studies (1), and further knowledge has come from x-ray crystal structures of acetylcholine binding proteins (AChBP), which are homologous to the extracellular domain of this receptor (2, 3). Cys-loop receptors are composed of five pseudo-symmetrically arranged subunits surrounding a central ion-conducting pore. Each subunit is composed of an extracellular, a transmembrane, and an intracellular domain. The extracellular domain (ECD) contains the ligand binding site, which is formed at the interface of two adjacent subunits by the convergence of three amino acid loops (A-C) from one (the principal) subunit and three ␤-strands (D-F) from the adjacent (or complementary) subunit. The transmembrane domain (TMD) contains four membrane spanning ␣-helices (M1-M4) and a short C terminus. M2 from each subunit lines the pore and contains regions responsible for channel gating and ion selectivity. A large loop between M3-M4 forms the intracellular domain and is involved in channel conductance and modulation.Structural details extrapolated from AChBP have greatly enhanced o...

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“…An important step in any process of structure determination is to assess the validity of the resulting structural models by testing their ability to predict the results of experiments that are not used as restraints. For this purpose, we used the structural ensembles for TS1 and TS2, obtained by using the restrained MD simulations, to predict the results of experimental double-mutant cycle measurements (⌽ IJ exp -values) (38)(39)(40)(41). The high level of agreement described below between the ⌽ IJ -values predicted from structures determined in the absence of such data and those measured experimentally is a stringent validation of the structural models presented in this work.…”

Section: Methodsmentioning

confidence: 70%

Determination of the folding transition states of barnase by using Φ_I-value-restrained simulations validated by double mutant Φ_IJ-values

Salvatella

Dobson

Fersht

et al. 2005

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

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The protein barnase folds from the denatured state into its native conformation via a high-energy intermediate. Using ⌽ I-values determined experimentally from single-point mutations as restraints in all-atom molecular dynamics simulations, we have determined ensembles of structures corresponding to the transition states for the formation of the folding intermediate and its conversion into the native state. We have also introduced a stringent validation of the approach used to calculate such structures by predicting interaction ⌽ IJ-values determined experimentally from a series of double-mutant cycles. The ensembles that we have obtained illustrate the heterogeneity in the nucleationcondensation process by which barnase folds. Obligatory steps of this process include the sequential formation of two folding nuclei, which correspond to the two main hydrophobic cores of the protein. Nonobligatory steps include the elongation of the strand ␤1 and the formation of the helix ␣2. The results confirm that the use of experimental observables such as ⌽I-values as restraints in molecular dynamics simulations is a powerful general strategy to characterize the relatively heterogeneous structural ensembles that populate nonnative regions of the energy landscapes of proteins.⌽-value ͉ protein folding ͉ restrained molecular dynamics simulations A detailed characterization of the energy landscapes of proteins is an extremely valuable tool for increasing our understanding of important biological processes such as protein folding and aggregation (1). Crucial information about the extent of formation of interresidue interactions in transition states for folding, the saddle points on the energy landscape of proteins, is provided by ⌽-values, parameters determined from a combination of kinetic measurements and protein engineering experiments (2, 3). The folding transition states of several small proteins have been characterized in detail by using the ⌽ I -value approach (4-9) and have revealed fundamental details about the mechanism of protein folding (10). The realization that ⌽ Ivalues bear the same relationship to molecular simulations of elusive states as do nuclear Overhauser effects to the structural determination of stable states by NMR spectroscopy has enabled transition states and folding pathways to be analyzed at atomic resolution (11)(12)(13)(14). Indeed, ⌽ I -values have been used directly as experimental restraints in computer simulations to calculate ensembles of structures representing the transition states of folding of several single-domain proteins, including acylphosphatase (12, 15), a fibronectin type III domain (16), two immunity proteins (17), and a series of SH3 domains (18). These studies have enabled important features of these ensembles to be recognized, including a description of the critical role of certain key residues in establishing the topology of the native state through a nucleation-condensation mechanism.In this work, we use ⌽ I -values as restraints in molecular dynamics (MD) simulations of a pro...

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A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase

Cited by 45 publications

References 53 publications

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Determination of the folding transition states of barnase by using Φ_I-value-restrained simulations validated by double mutant Φ_IJ-values

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A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase

Cited by 45 publications

References 53 publications

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Determination of the folding transition states of barnase by using ΦI-value-restrained simulations validated by double mutant ΦIJ-values

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Determination of the folding transition states of barnase by using Φ_I-value-restrained simulations validated by double mutant Φ_IJ-values