The partial charge of the nitrogen atom in peptide bonds.

A majority of the standard texts dealing with proteins portray the peptide link as a mixture of two resonance forms, in one of which the nitrogen atom has a positive charge. As a consequence, it is often believed that the nitrogen atom has a net positive charge. This is in apparent contradiction with the partial negative charge on the nitrogen that is used in force fields for molecular modeling. However, charges on resonance forms are best regarded as formal rather than actual charges and current evidence clearly favors a net negative charge for the nitrogen atom. In the course of the discussion, new ideas about the electronic structure of amides and the peptide bond are presented.     

Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule

Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.     

Far-infrared spectra of N-methylacetamide and related compounds and hydrogen-bond force constants

Far-infrared spectra of N-methylacetamide and other monosubstituted amides were measured in the liquid state in the region from 250 to 60 cm.^?1. Bands characteristic of the molecules with a peptide bond were found and assigned to the hydrogen-bond vibration and the C? N torsional vibration. Far-infrared and Raman spectra of crystalline N-methylacetamide were measured. The normal coordinate treatment for the low-temperature modification of cystalline N-methylacetamide was made. A reasonable set of force constants can explain all the spectra and their change due to the modification transition.     

Impact of cis-proline analogs on peptide conformation

The beta-turn is a common motif in both proteins and peptides and often a recognition site in protein interactions. A beta-turn of four sequential residues reverses the direction of the peptide chain and is classified by the phi and psi backbone torsional angles of residues i + 1 and i + 2. The type VI turn usually contains a proline with a cis-amide bond at residue i + 2. Cis-proline analogs that constrain the peptide to adopt a type VI turn led to peptidomimetics with enhanced activity or metabolic stability. To compare the impact of different analogs on amide cis-trans isomerism and peptide conformation, the conformational preference for the cis-amide bond and the type VI turn was investigated at the MP2/6-31+G** level of theory in water (polarizable continuum water model). Analogs stabilize the cis-amide conformations through different mechanisms: (1) 5-alkylproline, with bulky hydrocarbon substituent on the C(delta) of proline, increases the cis-amide population through steric hindrance between the alkyl substituent and the N-terminal residues; (2) oxaproline or thioproline, the oxazolidine- or thiazolidine-derived proline analog, favors interactions between the dipole of the heterocyclic ring and the preceding carbonyl oxygen; and (3) azaproline, containing a nitrogen atom in place of the C(alpha) of proline, prefers the cis-amide bond by lone-pair repulsion between the alpha-nitrogen and the preceding carbonyl oxygen. Preference for the cis conformation was augmented by combining different modifications within a single proline. Azaproline and its derivatives are most effective in stabilizing cis-amide bonds without introducing additional steric bulk to compromise receptor interactions.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

Analogues of intermediates in the action of pig kidney prolidase

Dicarboxylic acids, resembling the collected substrates for the reverse peptide bond forming reaction, were bound several orders of magnitude more tightly than substrates, products, or previously known competitive inhibitors of reactions catalyzed by pig kidney prolidase (EC 3.4.13.9), a dipeptidase that cleaves peptide bonds to the nitrogen atom of proline. Other inhibitors containing a phosphoryl or phosphonyl group in addition to a carboxyl substituent were bound even more tightly, in a manner consistent with their possible resemblance to tetrahedral intermediates in substrate hydrolysis. These included several analogues of phosphoenol pyruvate, of which the most potent was (Z)-3-bromophosphoenolpyruvate (Ki = 4.6 x 10(-9) M). Ki values were found to vary with changing pH in a manner consistent with displacement of a hydroxide ion from the active site.     

Investigating diproline segments in proteins: occurrences, conformation and classification

The covalent linkage between the side‐chain and the backbone nitrogen atom of proline leads to the formation of the five‐membered pyrrolidine ring and hence restriction of the backbone torsional angle ? to values of ?60 °± 30° for the L ‐proline. Diproline segments constitute a chain fragment with considerably reduced conformational choices. In the current study, the conformational states for the diproline segment ( ^L Pro‐ ^L Pro) found in proteins has been investigated with an emphasis on the cis and trans states for the Pro‐Pro peptide bond. The occurrence of diproline segments in turns and other secondary structures has been studied and compared to that of Xaa‐Pro‐Yaa segments in proteins which gives us a better understanding on the restriction imposed on other residues by the diproline segment and the single proline residue. The study indicates that P_II–P_II and P_II–α are the most favorable conformational states for the diproline segment. The analysis on Xaa‐Pro‐Yaa sequences reveals that the Xaa‐Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The present study may lead to a better understanding of the behavior of proline occurring in diproline segments which can facilitate various designed diproline‐based synthetic templates for biological and structural studies. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 54–64, 2012.     

Cis-trans isomerism of the peptide bond

The molecular orbital method PCILO is applied to eight. N-monsubstituted amides. Experimentally known geometric properties are reasonably predicted by minimization of total energy with respect to molecular geometry. The same procedure shows that molecular deformations during rotation around the peptide bond significantly lower calculated barriers. Experimental heats of activation and the free-energy changes associated with cis–trans isomerism are in good agreement with those calculated, which include qualitative estimates of configurational entropy contributions to the isomerism energies. Both the calculations and revised infrared data indicate that N-phenylurethane, which has been used as a model for the cis peptide bond, should be predominantly trans. However the variations in rotational barriers and cis–trans isomerism energies among the N-monosubstituted amides provide no reason to suppose that the cis peptide bond should be excluded from stable protein conformations.     

Effects of L-proline and some of its analogs on retinal spreading depression

L-Protine inhibits glumate-based spreading depressions (SDs) at low concentrations (2–2.5mM) and promotes K⁺-based SDs at higher concentrations (5 mM). The inhibition of glutamate-based SDs was postulate receptors on somatic and dendritic plasma membranes. The binding of proline to glutamate receptors was furthermore postulated to result in a result in a release of K⁺ from the intracellular compartment, enhancing the extracellular K⁺ concentration sufficiently to promote K⁺ based SDs. A proline analog, L-baikiain, containing a double bond and one more based A atom in the ring structure than proline had similar affects as the latter amino acid, but an analog, L-azetidine-2-hydroxylic acid, with one less C atom in the ring had little effect on SD in the retina.     

Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH

The molecular conformations of the linear oligopeptides H-(L -Ala)_n-L -Pro-OH, with n = 1,2 and 3, have been investigated. ¹³C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)_n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from ¹H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.     

13C multiplet nuclear magnetic resonance relaxation-derived ring puckering and backbone dynamics in proline-containing glycine-based peptides.

13CH2-multiplet nuclear magnetic resonance relaxation studies on proline (P)-containing glycine (G)-based peptides, GP, PG, GPG, PGG, and GPGG, provided numerous dipolar auto- and cross-correlation times for various motional model analyses of backbone and proline-ring bond rotations. Molecular dynamics simulations and bond rotation energy profiles were calculated to assess which motions could contribute most to observed relaxation phenomena. Results indicate that proline restricts backbone psi 1, psi 2, and phi 2 motions by 50% relative to those found for a polyglycine control peptide. psi 1 rotations are more restricted in the trans-proline isomer state than in the cis form. A two-state jump model best approximates proline ring puckering which in water could occur either by the C gamma endo-exo or by the C2 interconversion mechanism. The temperature dependence (5 degrees to 75 degrees C) of C beta, and C gamma, and C delta angular changes is rather flat, suggesting a near zero enthalpic contribution to the ring puckering process. In lower dielectric solvents, dimethylsulfoxide and methanol, which may mimic the hydrophobic environment within a protein, the endo-exo mechanism is preferred.     

Force-based analysis of multidimensional energy landscapes: application of dynamic force spectroscopy and steered molecular dynamics simulations to an antibody fragment-peptide complex

Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between Gly^H-H40 of the antibody fragment and Glu^Oε-6_peptide of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement.     

Influence of alkyl group on amide nitrogen atom on fluorescence quenching of tyrosine amide and N-acetyltyrosine amide

The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.     

The x ray structure of thiazolidine-4-carboxylic acid

The X ray crystal structure of thiazolidine-4-carboxylic acid has been determined. It appears that the anomalously low basicity of this compound in comparison with that of its non-sulfur-containing analogue, proline, is due to the resonance stabilization of the unprotonated form of thiazolidine-4-carboxylic acid. This stabilization is conferred through an increased interaction of the sulfur atom with the thiazolidine-4-carboxylic acid ring that is only possible when the nitrogen atom maintains a trigonal geometry. This effect seems also to account for the greater instability of N-hydroxymethylamines formed from thiazolidine-4-carboxylic acid as compared to those formed from proline. The crystal structure shows, in addition, an intramolecular hydrogen bond in combination with a bifurcated hydrogen bond.     

Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on‐column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3‐phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N² position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N² position on enantiomerization kinetics. However, methylation on N⁴ position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nmr and computer-aided studies of the three interchanging stereoisomers of the cyclic hexapeptide cyclo[-Pro1-Gly2-Glu3(OBzl)-Pro4-Phe5-Leu6-]: Unexpected observation of a cis isomer of a secondary amide peptide bond in the presence of two trans proline peptide bonds

The cyclic hexapeptide cyclo[-Pro¹-Gly²-Glu³(OBzl)-Pro⁴-Phe⁵-Leu⁶-] ( 1 ; OBzl: benzyl ester) was modeled and synthesized to be used as a chiral site for the separation of enantiomers. Total correlation spectroscopy and nuclear Ovehauser effect spectroscopy spectra of the peptide in CDCl₃ showed the presence of three stereoisomers. The two dominant stereoisomers 1a and 1b exchanged chemically with each other, while the minor stereoisomer 1c exchanged exclusively with the stereoisomer 1b . Stereoisomer 1a had two cis proline peptide bonds while stereoisomer 1b had all-trans peptide bonds. The stereoisomer 1c had, for nonstrained peptides, an unusual cis phenylalanine peptide bond while both proline peptide bonds were trans. © 1993 John Wiley & Sons, Inc.     

3D-QSAR studies of 2,2-diphenylpropionates to aid discovery of novel potent muscarinic antagonists

Muscarinic acetylcholine receptors (mAChRs) consisting of five known subtypes, are widely distributed in both central and peripheral nervous systems for regulation of a variety of critical functions. The present theoretical study describes correlations between experimental and calculated molecular properties of 15 α-substituted 2,2-diphenylpropionate antimuscarinics using quantum chemical and pharmacophore generation methods to characterize the drug mAChR properties and design new therapeutics. The calculated stereoelectronic properties, such as total energies, bond distances, valence angles, torsion angles, HOMO–LUMO energies, reactivity indices, vibrational frequencies of ether and carbonyl moieties, and nitrogen atom proton affinity were found to be well correlated when compared with experimentally determined inhibition constants from the literature using three muscarinic receptor assays: [³H]NMS receptor binding, α-amylase release from rat pancreas, and guinea pig ileum contraction. In silico predicted toxicity on rat oral LD₅₀ values correlated well with the [³H]NMS binding in N4TG1 cells and α-amylase release assays, but not the ileum contraction assay. Next, to explore the functional requirements for potent activity of the compounds, we developed a preliminary 3D pharmacophore model using the in silico techniques. The resulting model contained a hydrogen bond acceptor site on the carbonyl oxygen atom and a ring aromatic feature on one of the two aromatic rings in these compounds. This model was used as a template to search an in-house database for novel analogs. We found compounds equal in inhibition potency to atropine and, importantly, six not reported before as antimuscarinics. These results demonstrate that this QSAR approach not only provides a basis for understanding the molecular mechanism of action but a pharmacophore to aid in the discovery and design of novel potent muscarinic antagonists.     

Conformational characteristics of polypeptides containing isolated L-proline residues with cis peptide bonds

The conformational characteristics of the peptide sequence X-l-Pro, where X  Gly or l-Ala and the peptide bond joining X and l-Pro is cis, are evaluated. Semi-empirical potential functions are used to estimate the contributions to the conformational energy made by the non-bonded van der Waals' and electrostatic interactions and the intrinsic torsional potentials about the NC^a and C^aC′ bonds. Rotations φ₁ and ψ₁ about the NC^a and C^aC′ bonds in residue X and rotation ψ₂ about the C^aC′ bond in l-Pro are permitted, while the angle of rotation φ₂ about the NC^a bond in l-Pro is fixed at 120 ° by the pyrrolidine ring. The presence of the cis peptide bond connecting X and l-Pro renders the backbone rotations φ₁, ψ₁ in X dependent upon the rotation ψ₂ about the C^aC′ bond in l-Pro. (Interdependence of rotations in neighboring residues joined by a cis peptide bond was previously observed in l-alanine oligomers.) The number of energetically allowed conformations for the Gly and l-Ala residues preceding a cis peptide bond l-Pro residue are found to be substantially reduced from those permitted when the peptide bond is trans or when l-Pro is replaced by an amino acid residue. On the other hand, ψ₂ = 100 to 160 ° (cis′) and 300 to 0 ° (trans′) are found to be the lowest energy conformations of the l-Pro residue irrespective of the cis or trans conformation of the X-l-Pro peptide bond.     

Quantum chemistry analysis of the mechanism of action of proteolytic enzymes. I. The state of the cleavable bond of substrates and intermediates

Electronic parameters of amide and ester bonds in some compounds, modelling substrates of proteolytic enzymes, and electronic properties of corresponding tetrahedral compounds, which are intermediates of the hydrolytic reaction, were calculated by the CNDO/2 method. The nature of substituents and the formation of the hydrogen bond by the carbonyl oxygen atom were shown to have no sufficient influence on the charges and bond orders of the amide group. The dramatic dependence of the amide electronic state from the distort degree of its planar structure was found. The resonance stabilization was shown to be absent in the bicyclic beta-lactams. The pK alpha values of the amide nitrogen atom were calculated at various hybridization states in amides.