Electrostatic polarization is crucial for reproducing pKa shifts of carboxylic residues in Turkey ovomucoid third domain

We have computed pKa shifts for carboxylic residues of the serine protease inhibitor turkey ovomucoid third domain (residues Asp7, Glu10, Glu19, Asp27, and Glu43). Both polarizable and nonpolarizable empirical force fields were employed. Hydration was represented by the surface generalized Born and Poisson-Boltzmann continuum model. The calculations were carried out in the most physically straightforward fashion, by directly comparing energies of the protonated and deprotonated protein forms, without any additional parameter fitting or adjustment. Our studies have demonstrated that (i) the Poisson-Boltzmann solvation model is more than adequate in reproducing pKa shifts, most likely due to its intrinsically many-body formalism; (ii) explicit treatment of electrostatic polarization included in our polarizable force field (PFF) calculations appears to be crucial in reproducing the acidity constant shifts. The average error of the PFF results was found to be as low as 0.58 pKa units, with the best fixed-charges average deviation being 3.28 units. Therefore, the pKa shifts phenomena and the governing electrostatics are clearly many-body controlled in their intrinsic nature; (iii) our results confirm previously reported conclusions that pKa shifts for protein residues are controlled by the immediate environment of the residues in question, as opposed to long-range interactions in proteins. We are confident that our confirmation of the importance of explicit inclusion of polarization in empirical force fields for protein studies will be useful far beyond the immediate goal of accurate calculation of acidity constants.     

Effects of the residues on the excitation energies of protonated Schiff base of retinal (PSBR) in bR: a TD-DFT study

Effects of the residues on the excitation energies of protonated Schiff base of retinal (PSBR) in bacteriorhodopsin have been investigated by means of time-dependent density functional theory. The residues around PSBR are replaced by the point charges on atoms. The structures of PSBR and residues are referred from X-ray data. The atomic charges on the each residue were calculated the B3LYP/6-311G(d,p) level. The excitation energy of PSBR perturbed by the point charges on atoms of each residue was calculated at the B3LYP/6-31G(d,p) level. A total of 23 residues and five water molecules around PSBR were considered in the calculations. The large spectral shifts were caused by the Asp212 and Asp85. The origin of the spectral shifts was discussed on the basis of theoretical results.     

Large lanthanide-induced chemical shifts in cobalt complexes of enethials (α,β-unsaturated thioaldehydes)

Eu(fod)₃-, Yb(fod)₃- and Pr(fod)₃-induced chemical shifts of the ‘thioaldehydic’ protons in enethial ligands complexed to a cobalt cyclopentadienyl group are unusually large and in the same direction (10–30 ppm downfield per mole of shift reagent per mole of substrate). The shifts of the protons induced by Eu(fod)₃ and Pr(fod)₃ in the enethial ligands show an alternation in sign on proceeding away from the sulfur atom. In contrast to the results with the fod reagents, the ytterbium and lanthanum shift reagents Yb(thd)₃ and La(thd)₃ caused only small shifts of protons in the 2-phenylpropenethial ligand. No induced shifts with the Eu or Pr reagents were observed for a cyclopentadienyl cobalt complex of dithioglyoxal. The induced shifts in these enethial complexes may be caused by varying blends of complex formation, contact and pseudocontact shifts. Caution is advised in assigning origins to lanthanide induced shifts in such organometallic systems.     

Phosphopeptide elution times in reversed-phase liquid chromatography

Elution time shifts between 33 different peptides and their corresponding phosphopeptides ranging from 4 amino acid residues to 35 amino acids in length were systematically investigated using high-resolution reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analysis with trifluoroacetic acid as the ion pairing agent. Observed peptide elution time shifts for a single phosphorylation ranged from -5.28 min (for pYVPML) to +0.59 min (for HRDpSGLLDSLGR). Peptides containing a phosphotyrosine residue displayed a significant decrease in elution time following phosphorylation compared to their similar-sized peptides with phosphoserine or phosphothreonine residues. While peptide phosphorylation generally led to a decrease in the observed elution time, five peptides displayed increased elution times as a result of phosphorylation. For large peptides (> or =18 amino acids), the elution time shifts due to single phosphorylation were limited (ranging between -0.48 and +0.03 min), while the elution time shifts for small peptides (<18 amino acids) were characterized by a larger deviation (ranging between -5.28 and +0.59 min). The predictive capability for the observed RPLC elution time change due to phosphorylation has been suggested, which will aid in assigning confident phosphopeptide identifications and their subsequent confirmation.     

Selective chemical shift assignment of B800 and B850 bacteriochlorophylls in uniformly [13C,15N]-labeled light-harvesting complexes by solid-state NMR spectroscopy at ultra-high magnetic field

The electronic ground states of the bacteriochlorophyll a type B800 and type B850 in the light-harvesting 2 complex of Rhodopseudomonas acidophila strain 10050 have been characterized by magic angle spinning (MAS) dipolar (13)C-(13)C correlation NMR spectroscopy. Uniformly [(13)C,(15)N] enriched light-harvesting 2 (LH2) complexes were prepared biosynthetically, while [(13)C,(15)N]-B800 LH2 complexes were obtained after reconstitution of apoprotein with uniformly [(13)C,(15)N]-enriched bacteriochlorophyll cofactors. Extensive sets of isotropic (13)C NMR chemical shifts were obtained for each bacteriochlorin ring species in the LH2 protein. (13)C isotropic shifts in the protein have been compared to the corresponding shifts of monomeric BChl a dissolved in acetone-d(6). Density functional theory calculations were performed to estimate ring current effects induced by adjacent cofactors. By correction for the ring current shifts, the (13)C shift effects due to the interactions with the protein matrix were resolved. The chemical shift changes provide a clear evidence for a global electronic effect on the B800 and B850 macrocycles, which is attributed to the dielectrics of the protein environment, in contrast with local effects due to interaction with specific amino acid residues. Considerable shifts of -6.2 < Deltasigma < +5.8 ppm are detected for (13)C nuclei in both the B800 and the B850 bacteriochlorin rings. Because the shift effects for the B800 and B850 are similar, the polarization of the electronic ground states induced by the protein environment is comparable for both cofactors and corresponds with a red shift of approximately 30 nm relative to the monomeric BChl dissolved in acetone-d(6). The electronic coupling between the B850 cofactors due to macrocycle overlap is the predominant mechanism behind the additional red shift in the B850.     

Total enantioselectivity in the DNA binding of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [bpm = 2,2'-bipyrimidine; Me2bpy = 4,4'-dimethyl-2,2'-bipyridine

The binding of the three stereoisomers (DeltaDelta-, LambdaLambda- and DeltaLambda-) of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to a tridecanucleotide containing a single adenine bulge has been studied by 1H NMR spectroscopy. The addition of the DeltaDelta-isomer to d(CCGAGAATTCCGG)2 induced significant chemical shift changes for the base and sugar resonances of the residues at the bulge site (G3A4G5/C11C10), whereas small shifts were observed upon addition of the enantiomeric LambdaLambda-form. NOESY spectra of the tridecanucleotide bound with the DeltaDelta-isomer revealed intermolecular NOE's between the metal complex and the nucleotide residues at the bulge site, while only weak NOE's were observed to terminal residues to the LambdaLambda-form. Competitive binding studies were performed where both enantiomers were simultaneously added to the tridecanucleotide, and for all ratios of the two stereoisomers the DeltaDelta-isomer remained selectively bound at the bulge site with the LambdaLambda-enantiomer localised at the terminal regions of the tridecanucleotide. The meso-diastereoisomer (DeltaLambda) was found to bind to the tridecanucleotide with characteristics intermediate between the DeltaDelta- and LambdaLambda-enantiomers of the rac form. Two distinct sets of metal complex resonances were observed, with one set having essentially the same shift as the free metal complex, whilst the other set of resonances exhibited significant shifts. The NOE data indicated that the meso-diastereoisomer does not bind as selectively as the DeltaDelta-isomer, with NOE's observed to a greater number of nucleotide residues compared to the DeltaDelta-form. This study provides a rare example of total enantioselectivity in the binding of an inert transition metal complex to DNA, produced by the shape recognition of both ruthenium(II) centres.     

Identification of 4,5-asymmetrically substituted 1-(N-isoimido)-1,2,3-triazoles by benzene induced shifts in NMR spectra—VII

Proton chemical shifts, as well as solvent shifts induced by benzene, can be used for the structure determination of 4,5-asymmetrically substituted 1-(N-isoimido)-1,2,3-triazoles, derived from the oxidation of α-diketone bisaroylhydrazones. The geometry of the postulated ‘collision complex’ is discussed and an attempt is made to estimate theoretically the induced shifts of some of the protons.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

An investigation of complexes of some model tripeptides containing phenylalanine and tyrosine residues with DNA by Fourier1H NMR spectroscopy

Complexes of native and denatured DNA with model tripeptides containing phenylalanine or tyrosine residues flanked by lysine or arginine residues, respectively have been investigated by pulsed Fourier¹H NMR spectroscopy. The existence of shifts into the strong-field region of the signals of aromatic protons of the model tripeptides in the complexes both with native and with denatured DNA has been shown. Results have been obtained that indicate the possibility of the intercalation of the side chains of aromatic amino acid residues into the DNA double helix.All-Union Scientific-Research Institute of the Genetics and Breeding of Industrial Microorganisms, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 751–755, November–December, 1986.     

Nuclear Magnetic Resonance Studies. IV. Proton Chemical Shifts and Coupling Constants of 2-Norbornanols and Methyl-Substituted 2-Norbornanols

The proton magnetic resonance spectra of the two isomeric 2-norbornanols (I and II) and of eighteen of their methyl-substituted derivatives (III to XX) under the influence of tris(dipivalomethanato)europium, Eu(dpm)₃, have been measured. In all cases linear relationships between the observed shifts and the molar ratios of Eu(dpm)₃ to substrate were realized. The chemical shifts obtained from the extrapolation to zero concentration of Eu(dpm)₃ are in good agreement with the observed values. The coupling constants were found to be essentially unaffected by Eu(dpm)₃. Consequently, determination of a chemical shift by extrapolation or of a coupling constant from the shifted spectrum appears to be justified. Reassignments of a number of signals are discussed. Chemical shifts, normalized induced shifts, and coupling constants are tabulated.     

Lanthanide induced paramagnetic shifts—derivation of equilibrium constants and absolute shifts in Eu(DPM)3 systems

Relationships between the magnitude of induced ¹H NMR shifts and the equilibrium conditions in solutions containing lanthanide shift reagents are discussed and applied to the shifts induced in several organic molecules by Eu(DPM)₃. Evidence that monoadduct formation is the predominant equilibrium in these solutions, and values of absolute shifts and equilibrium constants are reported. The results indicate that a contact mechanism makes the major contribution to the observed shifts in 4-methylpyridine. In the case of 2,4,6-trimethylpyridine the observed shifts are largely pseudocontact in origin.     

Influence of NMR Data Completeness on Structure Determinations of Homodimeric Proteins

The structure determination of homodimeric proteins by NMR using conventional NOESY experiments is still challenging due to the degeneracy of the chemical shifts in the identical monomers, which causes ambiguity in the NOE assignments. Residues involved in the interface between two monomers provide essential intermolecular NOEs for the structure determinations of homodimeric proteins. Hence NMR data, such as NOE peak lists and chemical shift assignments of these interface residues, play a crucial role for the successful structure determination of homodimeric proteins. This paper extends our previous report (Lin, Y.‐J.; Kirchner, D. K.; Güntert, P. J. Magn. Reson.­ 2012 , 222, 96) and investigates the influence of incomplete NOESY peak lists combined with incomplete ¹H chemical shift assignments of the interface residues on the structure determination of homodimeric proteins using the program CYANA. Data incompleteness was simulated by random omission of both NOESY cross peaks and interface ¹H chemical shifts. Our results for three proteins with different percentages of interface residues reveal that the algorithm can tolerate about 40–50% NOESY peak omission with complete interface chemical shift assignments, which indicates that partial NOESY peak omission does not cause severe problems when the interface chemical shifts are completely assigned. Combining NOESY peak omission with incomplete interface chemical shift assignments, the tolerance for interface chemical shift omission decreases with the extent of omitted NOESY peaks. The tolerance for unassigned interface side chain, methyl and aromatic chemical shifts is affected more strongly by NOESY peak omission than that for the omission of general interface ¹H chemical shifts including the backbone. In general about 10–30% peaks omission is tolerated in conjunction with missing chemical shift assignments. If more NOESY peaks are omitted calculations gradually become unstable and tend not to tolerate any missing interface chemical shifts. A large amount of omitted NOESY peaks, for instance 30% omission in our calculations, could decrease the tolerance for missing aromatic or methyl interface ¹H chemical shifts to as few as 2–4 missing chemical shifts, suggesting that complete aromatic and methyl ¹H chemical shift assignments are important when the NOESY peak data is significantly incomplete. Finally, for homodimeric proteins with a low percentage of interface residues, our results reveal that the omission of NOESY peaks, even at an extent of only 10%, can result in no tolerance against the omission of interface ¹H chemical shifts, suggesting that the completeness of both interface ¹H chemical shift assignments and NOESY peaks are important for the successful structure determination of proteins with a small homodimer interface.     

An investigation of complexes of some model tripeptides containing phenylalanine and tyrosine residues with DNA by Fourier1H NMR spectroscopy

Complexes of native and denatured DNA with model tripeptides containing phenylalanine or tyrosine residues flanked by lysine or arginine residues, respectively have been investigated by pulsed Fourier¹H NMR spectroscopy. The existence of shifts into the strong-field region of the signals of aromatic protons of the model tripeptides in the complexes both with native and with denatured DNA has been shown. Results have been obtained that indicate the possibility of the intercalation of the side chains of aromatic amino acid residues into the DNA double helix.     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

Hydrogen-bonding effect on C NMR chemical shifts of amino acid residue carbonyl carbons of some peptides in the crystalline state

¹³C cross polarization-magic angle spinning NMR spectra were measured for a series of peptides containing -valine, -leucine and -aspartic acid residues, for which the crystal structures were already determined by X-ray diffraction, in order to investigate the relationship between hydrogen-bond lengths (R_N…O) and ¹³C chemical shifts of amide carbonyl carbons in the peptides. From these experimental results, it was found that the isotropic ¹³C chemical shifts (δ_iso) of the amino acid residues move linearly downfield with a decrease in R_N…O within the hydrogen-bonded length range considered here and also shown in our previous work on glycine and -alanine residues as expressed by δ_iso(ppm) = a − bR_N…O(Å) where a and b are 215.4 (ppm) and 14.2 (ppm Å⁻¹) for the -valine residue, 202.2 (ppm) and 10.0 (ppm Å⁻¹) for the -leucine residue, and 199.0 (ppm) and 9.6 (ppm Å⁻¹) for the -aspartic acid residue, respectively. Using these relations, the R_N…O values of some polypeptides in the crystalline state were determined through the observation of the amide carbonyl carbon chemical shifts. These values were compared with those determined by the X-ray diffraction method. Furthermore, quantum-chemical calculation of the ¹³C shielding constant for a model compound was carried out by the finite perturbation theory INDO method in order to ascertain the ¹³C shielding behavior in the formation of hydrogen bonds.     

Oligomers of a polyester network

Oligomers of glycerol and succinic acid have been prepared by three different methods and characterised by solution ¹³C-NMR spectroscopy at 125.8 MHz. The first series of five oligomers was prepared by reacting glycerol with succinic anhydride; only one acid function of each succinic acid residue formed an ester by this means. They were readily distinguished as the shifts of their glycerol residues were dispersed over 16 ppm, and their shifts provided a guide to the assignment of shifts in the more elaborate oligomers. The second set of oligomers was prepared by treating glycerol with a small quantity of succinic acid, ester links being promoted by means of the reagent dicyclohexylcarbodiimide (DCCDI). When we used 2 mol of DCCDI/mol of acid in the presence of an excess of glycerol, no free acid functions remained, and a new set of oligomers was obtained. Furthermore, within this set the proportion of ring molecules was enhanced by repeating the reaction under much more dilute conditions. In this way the shifts of two ring oligomers were recognized in the spectrum. A different set of oligomers again was obtained when the esterification was performed with 1 mol of DCCDI/mol of succininic acid. After the first generation of oligomers had been identified in the mixtures produced by these three experiments, the reactions continued to produce larger oligomers with new fine structure features in the spectra. Glycerol trisuccinate was prepared in a pure form and heated in a vacuum to eliminate succinic acid groups and allow the formation of oligomers with two and three branch points. The structures successively produced by this reaction were readily recognized. In all we were able to recognize the formation of component structures in at least 17 different oligomers. The shifts of the carbons of the glycerol residues are sensitive to the substitution pattern at that residue, to whether the succinic acid residue to which they are linked has reacted a second time, and in some cases the methine shift is sensitive to how the succinic acid residue attached to a neighboring methylene carbon has reacted. © 1994 John Wiley & Sons, Inc.     

Cellulose Iβ investigated by IR-spectroscopy at low temperatures

Highly crystalline oriented Halocynthia roretzi cellulose Iβ films were investigated by IR-spectroscopy between ?180 and +10 °C. Changes in the IR-spectra induced by temperature were compared to published changes induced by mechanical stretching. This made it possible to conclude that frequency shifts in the O–H stretching region of the IR-spectra due to temperature were not predominantly an indirect effect of thermal expansion leading to greater O–O distances, but were due directly to the effect of temperature on the O–H···O hydrogen bonds. Temperature induced frequency shifts of C–H stretching bands were consistent with the presence of weak inter-sheet C–H···O bonds. Furthermore, no phase transition in cellulose Iβ was found between ?180 and +10 °C.     

Structural determinations of some chloroazepine-2,5-diones using a lanthanide shift reagent

The use of a lanthanide shift reagent, Eu(fod)₃, to aid in the structural assignments of some chloroazepine-2,5-diones is described. The chloroazepine-2,5-diones, synthesized via the Schmidt reactions of chloro-1,4-benzoquinones, could not readily have their structures assigned by other spectroscopic methods. Correlations of plotted lanthanide induced shifts in pmr studies demonstrated that there was a large positional dependence on the magnitude of induced shifts. The large difference in the magnitude of induced shifts made it possible to assign protons and methyl substituents to specific positions on the azepine ring, thus assigning the structure of the compound.     

A solid state 13C NMR, crystallographic, and quantum chemical investigation of chemical shifts and hydrogen bonding in histidine dipeptides

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.     

Direct test of the Gaussian-chain model for treating residual charge-charge interactions in the unfolded state of proteins

The Gaussian-chain model for treating residual charge-charge interactions was critically tested by recent experimental pK(a) results for individual Asp, Glu, and His residues in the unfolded drkN SH3 domain. Predicted pK(a)'s were in good agreement with experiment. The clustering of Asp and Glu residues along the sequence was suggested to limit pK(a) shifts and contribute to the folding stability by destabilizing the unfolded state.