Conformational dependence of <Superscript>13</Superscript>C shielding and coupling constants for methionine methyl groups

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of ³ J _CSCC, ³ J _CSCH, and the isotropic shielding, σ_iso. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-¹³C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, ³ J _CSCC/³ J _COCC ~ 0.7. It is further demonstrated that the ³ J _CSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ¹³Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large ~100° χ³ values. The utility of the δ¹³Cε as a basis for estimating the gauche/trans ratio for χ³ is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.     

Conformational dependence of the vicinal proton coupling constant for the Cα?Cβ bond in peptides

We use the nmr data concerning the C^αH? C^βH fragment in eight peptides with rigid side chains to parametrize a Karplus correlation between the vicinal proton J_αβ coupling constant and the dihedral angle θ. When considering molecules containing the fragment C^αH^α? C^βH^βH^β′, the three-dimensional structure of the model peptides does not need to be known with accurate precision, since each set of J_αβ and J_αβ′ coupling constants is then related to the coefficients of the Karplus equation. A good correlation is observed with the Karplus equation, which is in substantial agreement with the J_αβ coupling constants reported for rigid as well as rotating C^α? C^β bonds in peptides.     

HNCO-based measurement of one-bond amide <Superscript>15</Superscript>N-<Superscript>1</Superscript>H couplings with optimized precision

A pair of 3D HNCO-based experiments have been developed with the aim of optimizing the precision of measurement of ¹J_NH couplings. Both pulse sequences record ¹J_NH coupling evolution during the entire constant time interval that ¹⁵N magnetization is dephasing or rephasing with respect to the directly bonded ¹³C′ nucleus, with ¹⁵N¹³C′ multiple quantum coherence maintained during the ¹³C′ evolution period. The first experiment, designed for smaller proteins, produces an apparent doubling of the ¹J_NH coupling without any accompanying increases in line width. The second experiment is a J-scaled TROSY-HNCO experiment in which the ¹J_NH coupling is measured by frequency difference between resonances offset symmetrically about the position of the downfield component of the ¹⁵N doublet (i.e. the TROSY resonance). This experiment delivers significant gains in precision of ¹J_NH coupling measurement compared to existing J-scaled TROSY-HNCO experiments. With the proper choice of acquisition parameters and sufficient sensitivity to acquire a 3D TROSY-HNCO experiment, it is shown that ¹J_NH couplings can be measured with a precision which approaches or exceeds the precision of measurement with which the frequency of the TROSY resonance itself can be determined.     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Theoretical study of isotope effects on the stereodynamics of H<Superscript>+</Superscript>+HD and its isotopic variant D<Superscript>+</Superscript>+HD

The stereodynamics for H⁺+HD and its isotopic variant D⁺+HD were studied with a quasi-classical trajectory (QCT) method at a collision energy of 0.7 eV on the ground 1¹A′ potential energy surface (PES). The polarization-dependent differential cross-sections (PDDCSs) in the center-of-mass frame are presented here. Furthermore, the distribution of the angle between k and j′, p(θ _r ) and the distribution of the dihedral angle p(ϕ _r ) were calculated and are discussed. The results indicate that isotopic substitution exerts substantial effects on the differential cross-section and the product’s rotational polarization.     

Quantitative f torsion angle analysis in Desulfovibrio vulgaris flavodoxin based on six f related 3 J couplings

Quantitative φ-dihedral angle determinations of non-glycine and non-proline residues in Desulfovibrio vulgaris flavodoxin are carried out on the exclusive basis of ³ J coupling constants. In total 124 ³ JHNH _α , 123 ³ JHNC _′i , 118 ³ JHNC _β , 117 ³ JC′ _i–1Hα , 109 ³ JC′ _i–1C′i , and 103 ³ JC′ _i–1Hβ values form the experimental basis for translating J coupling data into geometry information using various combinations of Karplus parameters given in the literature. In addition, each backbone torsional angle φ is adjusted assuming different models of local geometry, either a rigid torsion, a Gaussian distribution centered at a distinct angle, or a two-site jump model. Numerical optimization is followed by a statistical significance evaluation to assess the results. It is found that experimental coupling constants of most of the residues involved in secondary structure elements agree best with those predicted from rigid local conformations. For dihedral angles in loop regions, mobility effects are not negligible, and a single torsion (Glu 42) is likely to adopt two distinct adjustments. However, α-helix conformations with –60° < φ < –45° give rise to an alternate solution with φ≈+170° with similar statistical significance when using the four traditionally determined proton-involved ³ J couplings. This ambiguity is efficiently avoided only when taking advantage of the complete data set comprising six available experimental ³ J coupling constants and of the degeneracy intrinsic to the Karplus relation. The optimized φ conformations are compared with reference values from the crystal structure of flavodoxin.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Applications of Variable-angle Sample Spinning Experiments to the Measurement of Scaled Residual Dipolar Couplings and <Superscript>15</Superscript>N CSA in Soluble Proteins

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 32% w/v have been recorded at 700 MHz under sample spinning conditions at the magic angle (54.7°) and at an angle of 45.5°. At the magic angle, the ¹H–¹⁵N HSQC spectrum of ubiquitin in bicelles is virtually indistinguishable from the one recorded on the protein in solution. Spinning the sample at the magic angle creates an isotropic environment with no preferred bicelle orientations, thus allowing the determination of scalar coupling constants. For an angle of rotation of 45.5°, the bicelles orient with their normal perpendicular to the spinning axis leading to the observation of strong residual dipolar couplings and chemical shift variations of the ¹⁵N resonances. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

<Superscript>31</Superscript>P NMR correlation maps of <Superscript>18</Superscript>O/<Superscript>16</Superscript>O chemical shift isotopic effects for phosphometabolite labeling studies

Intramolecular correlations among the ¹⁸O-labels of metabolic oligophosphates, mapped by J-decoupled ³¹P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the ¹⁸O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of ¹⁸O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled ³¹P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally ¹⁸O labeled in perfused mammalian hearts.     

Broadband <Superscript>15</Superscript>N–<Superscript>13</Superscript>C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient RN_n^{n_S, n_k} {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} symmetry-based dual channel RF pulse schemes for γ-encoded broadband ¹⁵N–¹³C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and ¹⁵N–¹³C chemical shift correlation experiments. Employing such ¹³C–¹⁵N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.     

A dihedral angle-vicinal proton coupling constant correlation for the α–β bond of amino acid residues

A coupling constant-dihedral angle correlation for the H? Cα? Cβ? H system of amino acid residues in peptides has been derived from a set of model compounds covering the full range of dihedral angles. The expression obtained, J = 11.0 cos₂ θ ?1.4 cos θ + 1.6 sin₂θ, is close to those already used in pmr studies of peptide conformation, and provides a firmer foundation for them. A factor limiting the precision of this and other “Karplus relations” is illustrated.     

Effects of Ce<Superscript>3+</Superscript> on Conformation and Activity of Superoxide Dismutase

Ce³⁺ in various concentrations was added to superoxide dismutase (SOD) from rat eryhrocyte in vitro to gain insight into the mechanism of molecular interactions between Ce³⁺ and SOD. The results showed that the reaction between SOD and Ce³ was two order, which meant that the SOD activity was markedly accelerated by a low concentration of Ce³⁺ and inhibited by a high concentration of Ce³⁺. The spectroscopic assays suggested that the Ce³⁺ was determined to directly bind to SOD; the binding site of Ce³⁺ to SOD was 0.96, and the binding constants (K _A) were 6.78 × 10⁵ and 1.68 × 10⁵L·mol⁻¹; the binding Ce³⁺ entirely altered the secondary structure of SOD. It implied that the Ce³⁺ coordination created a new metal ion-active site form in SOD, thus leading to an enhancement in SOD activity.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Asymmetry of <Superscript>13</Superscript>C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

Deuterium isotope effects on <Superscript>15</Superscript>N backbone chemical shifts in proteins

Quantum mechanical calculations are presented that predict that one-bond deuterium isotope effects on the ¹⁵N chemical shift of backbone amides of proteins, ¹Δ¹⁵N(D), are sensitive to backbone conformation and hydrogen bonding. A quantitative empirical model for ¹Δ¹⁵N(D) including the backbone dihedral angles, Φ and Ψ, and the hydrogen bonding geometry is presented for glycine and amino acid residues with aliphatic side chains. The effect of hydrogen bonding is rationalized in part as an electric-field effect on the first derivative of the nuclear shielding with respect to N–H bond length. Another contributing factor is the effect of increased anharmonicity of the N–H stretching vibrational state upon hydrogen bonding, which results in an altered N–H/N–D equilibrium bond length ratio. The N–H stretching anharmonicity contribution falls off with the cosine of the N–H···O bond angle. For residues with uncharged side chains a very good prediction of isotope effects can be made. Thus, for proteins with known secondary structures, ¹Δ¹⁵N(D) can provide insights into hydrogen bonding geometries. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Program MULDER -- a tool for extracting torsion angles from NMR data

MULDER (Mostly UniversaL Dihedral angle ExtractoR) is a program for extraction of torsion angle information from NMR data. Currently, it can analyze two types of input data: The torsion angle data, where several ³J-coupling constants and/or interatomic distances are combined in order to reduce the torsion angle ambiguity arising from solving the isolated Karplus (or distance) equation, and the sugar pucker data, where the dynamics of five-membered sugar rings is evaluated by postprocessing the results calculated from ³J_(HH) coupling constants by program PSEUROT. Program MULDER can be used either as an alternative to r-MD programs in situations where only specific structural features are studied, or as a preparatory tool in connection with full r-MD structure calculation for extraction of unambiguous torsion angle restraints.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Computational study of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Sodium proton antiporters are ubiquitous membrane proteins that catalyze the exchange of Na⁺ for protons throughout the biological world. The Escherichia coli NhaA is the archetypal Na⁺/H⁺ antiporter and is absolutely essential for survival in high salt concentrations under alkaline conditions. Its crystal structure, accompanied by extensive molecular dynamics simulations, have provided an atomically detailed model of its mechanism. In this study, we utilized a combination of computational methodologies in order to construct a structural model for the Na⁺/H⁺ antiporter from the gram-negative bacterium Vibrio parahaemolyticus. We explored its overall architecture by computational means and validated its stability and robustness. This protein belongs to a novel group of NhaA proteins that transports not only Na⁺ and Li⁺ as substrate ions, but K⁺ as well, and was also found to miss a β-hairpin segment prevalent in other homologs of the Bacteria domain. We propose, for the first time, a structure of a prototype model of a β-hairpin-less NhaA that is selective to K⁺. Better understanding of the Vibrio parahaemolyticus NhaA structure-function may assist in studies on ion transport, pH regulation and designing selective blockers.