Carbon-13 nuclear magnetic resonance titration shifts in amino acids

Quirt, A. R.; Lyyerla, J. R.; Peat, I. R.; Cohen, Jack S.; Reynolds, W. F.; Freedman, M. H.

doi:10.1021/ja00809a038

Cited by 131 publications

(71 citation statements)

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“…The peak was in an unusual position for an Asp-Glu side chain, exhibiting an upfield chemical shift to 174 ppm in its Cδ resonance (Fig. 4), far from the typical Cδ resonances of surface Glu residues, which range from 178 to 185 ppm depending on whether the group is neutral or charged, respectively (16,23). The Cγ resonance was ∼31.8 ppm, near the upfield limit of the normal range for Glu residues from 33 to 39 ppm.…”

Section: Resultsmentioning

confidence: 91%

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Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

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

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An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK a values near 7. When buried together their pK a values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.electrostatics | internal charge | low-barrier hydrogen bond | dielectric constant | enzyme design P aired ionizable groups buried in hydrophobic environments inside proteins are an uncommon but essential structural motif necessary for H + transport (1), e − transfer (2), catalysis (3-5), and other important biochemical processes. Despite their importance, the properties of these buried ionizable pairs are poorly understood because they are difficult to study experimentally in the proteins where they play functional roles. When the pair consists of a basic and an acidic group it is usually assumed to be charged, but this is often just speculation without direct experimental support (6, 7). In principle, a Coulomb interaction between two ionizable groups of opposite polarity buried in a dehydrated environment inside a protein can be very strong, but this has not been established experimentally. Simulations suggest that buried ion pairs are always destabilizing (8); however, from an experimental perspective it is not known if the favorable Coulomb interaction would be sufficient to compensate for the unfavorable dehydration of the two buried charges or if the net effect of the pair on the thermodynamic stability of a protein would be stabilizing or not. These issues were examined in detail with an artificial ion pair buried in the hydrophobic interior of staphylococcal nuclease (SNase).The buried ion pair was engineered by introducing the V23E and L36K substitutions in a highly stable form of SNase. The pK a values of Glu-23 and Lys-36 introduced individually are anomalous (pK a of 7.1 for ref. 9; ref. 10), consistent with the groups existing in hydrophobic environments that are neither as polar nor as polarizable as water (11-13). Structural modeli...

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Section: Resultsmentioning

confidence: 91%

“…Side chain atoms for Glu-23 and Lys-36 in the Δ+PHS/V23E/L36K variant are shown as balls-and-sticks. chemical shift change of the Cδ of a titrating Glu residue, which ranges from 3 to 5 ppm (23). Small shifts of the order of ∼1 ppm usually result from the titration of another ionizable group coupled to the residue being monitored (16).…”

Section: Resultsmentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

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

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“…changes in the chemical shifts of C,, and C p J upon amino protonation (26,27). Also: the smaller value of 'J(c,, ,Ha,), 129.6 Hz, compared to 'J(CpJ ,Hp,), 145.5 Hz, is consistent with our assignment since CH3NH3', 'J(C,H) = 145 Hz (28), and in CH3CONH2, 'J(C,H) = 129 Hz (29).…”

Section: Assignment Of 13c and 'H Resonances Of The P-alanyl Groupmentioning

confidence: 99%

A ¹H and ¹³C nuclear magnetic resonance study of carnosine

Friedrich¹,

Wasylishen²

1986

Can. J. Chem.

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The proton and carbon-13 resonance signals of carnosine (β-alanyl-L-histidine) were unambiguously assigned using a variety of nmr techniques including proton–carbon chemical shift correlations, titrations of nmr chemical shifts, coupling constants, and isotope shifts. From the 13C nmr titration, carnosine's three pKa values were estimated to be 2.7, 7.1, and 10.6, and it was found that the imidazole ring existed predominantly as the 3-H tautomer in basic solution. Conformational information about the Cα—Cβ bond and about the N—Cα bond was deduced from observed 3J(C,H) and 3J(H,H) values. The 13C nmr spectrum of carnosine in solution is also compared with that obtained for a solid sample.

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“…The 'state of the art' in the prediction of chemical shifts of amino acids is such that molecular orbital calculations do not satisfactorily account for 13C shift changes on titration (2). Until such time that theory can satisfactorily account for such changes, can be extended to predict chemical shifts as well as these changes, and can be easily applied, empirical methods of assigning chemical shifts by addition of parameter terms will be widely used.…”

Section: Resultsmentioning

confidence: 99%

“…The 13C spectra of amino acids are useful not only for studying their properties (1)(2)(3), but also as a starting point for the structural deterinination of con~pounds derived from ainino acids, such as peptides and proteins (4)(5)(6)(7), and even complex pyrazinones such as arglecin, deoxyaspergillic acid, and flavacol (8). One illethod used to assign 13C spectra of amino acids was that using additive parameter terms (9) which, however, were derived from studies on the chemical shifts of entirely different compounds.…”

Section: Introductionmentioning

confidence: 99%

Empirical equations for predicting ¹³C chemical shifts in nuclear magnetic resonance spectra of certain types of amino acids

MacDonald¹,

Bishop²,

Mazurek³

1976

Can. J. Chem.

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13C chemical shift data were obtained for amino acids related to glycine which had the following types of substituents on C-2: alkyl, branched chain alkyl, ω-amino alkyl, and ω-guanido alkyl. Such data were obtained for aqueous solutions at a pH between the pK values of the carboxyl and 2-amino groups (zwitterion form). The data were subjected to graphical and statistical analysis to obtain separate predictive equations for C-1, C-2, and other carbon atoms. Data for the change in chemical shifts on titration with acid were also obtained to aid in 13C assignments and to elucidate configuration.

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Carbon-13 nuclear magnetic resonance titration shifts in amino acids

Cited by 131 publications

References 1 publication

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

A ¹H and ¹³C nuclear magnetic resonance study of carnosine

Empirical equations for predicting ¹³C chemical shifts in nuclear magnetic resonance spectra of certain types of amino acids

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Carbon-13 nuclear magnetic resonance titration shifts in amino acids

Cited by 131 publications

References 1 publication

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

A 1H and 13C nuclear magnetic resonance study of carnosine

Empirical equations for predicting 13C chemical shifts in nuclear magnetic resonance spectra of certain types of amino acids

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A ¹H and ¹³C nuclear magnetic resonance study of carnosine

Empirical equations for predicting ¹³C chemical shifts in nuclear magnetic resonance spectra of certain types of amino acids