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PubMed1993

Protein response to photodissociation of CO from carbonmonoxymyoglobin probed by time-resolved infrared spectroscopy of the amide I band.

Protein conformational changes coupled to the ligation reactions of carbon monoxide in myoglobin (Mb) are detected by time-resolved infrared spectroscopy. An apparatus based on a tunable diode laser operating in the region of 1650 cm-1 is used to probe changes in the amide I absorption band of the protein in response to photodissociation and subsequent rebinding of CO. The time course of changes in the amide I band is shown to follow the recombination of photolyzed CO with Mb. A time-resolved difference spectrum in the amide I region is generated by tuning the diode laser probe source. The features in the IR difference spectrum are assigned to the motions of the polypeptide backbone associated with the global relaxation of the protein from the ligated to the deoxy conformation. A static difference spectrum generated by subtracting FTIR spectra of carbonmonoxy-Mb and deoxy-Mb is essentially identical to the transient spectrum, indicating that the protein relaxation is complete with the 100-ns time resolution of the experiment.

Amides↗
PubMed1992

Redox-dependent changes in beta-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra.

The redox-dependent changes in secondary structure of cytochromes c from horse, cow, and dog hearts in water at 20 degrees C have been determined by amide I infrared spectroscopy. Second derivative amide I spectra were obtained by use of a procedure that includes a convenient method for the effective subtraction of the spectrum of water vapor in the system. The band at 1657 cm-1 representing the helix structure was unaffected by a change in redox state whereas changes in bands due to turns at 1680, 1672, and 1666 cm-1, unordered structure at 1650 cm-1, and beta-structures at 1632 and 1627 cm-1 occurred. About one-fourth of the beta-extended chain spectral region and one-fifth of the beta-turn region (involving a total of approximately 9-13 residues) were sensitive to the oxidation state of heme iron. No significant changes in the secondary structure of either the reduced or oxidized protein due to changes in ionic strength were detected. The localized structural rearrangements triggered by the changes in oxidation state of heme iron are consistent with differences in the binding of heme iron to a histidine imidazole nitrogen and a methionine sulfur atom from the beta-extended chain. The demonstrated ability to obtain highly reproducible second derivative amide I infrared spectra confirms the unique utility of such spectral measurements for localization of subtle changes in secondary structure within a protein, especially for changes among the multiple turns and beta-structures.

Amides↗
PubMed1994

Structure of glucagon-like peptide (7-36) amide in a dodecylphosphocholine micelle as determined by 2D NMR.

We have used 2D 1H NMR to determine the structure of glucagon-like peptide-1-(7-36) amide bound to a dodecylphosphocholine micelle. In this membranelike environment, the peptide hormone is shown to have a structure similar to that observed for glucagon. It consists of an N-terminal random coil segment (residues 1-7), two helical segments (7-14 and 18-29), and a linker region (15-17). The C-terminal helix is more stable than the N-helix as determined by amide proton exchange experiments. The C-terminal helix shows much larger alpha and amide proton upfield secondary shifts relative expected for a random coil conformation. This suggests a highly helical structure in this portion of the molecule. The C-terminal helix also has a much larger fraction of residues that are hydrophobic, presumably enhancing the interaction of this portion of the peptide with the micelle (or membrane). The structure refined from the NOESY data is not a uniform alpha-helix throughout residues 6-30. A uniform helix would not be perfectly amphiphilic since the hydrophobic face of the N-terminal portion of the helix is positioned in nearly perfect opposition to the hydrophobic face of the C-terminal portion. However, helical distortion around residues 15-17 allows a phase shift of the two helical segments to position nearly all of the hydrophobic residues (and none of the hydrophilic ones) on a single face of the distorted single helix as would be required to favorably interact with the hydrophobic portion of the micelle or membrane.

Amides↗
PubMed2000

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis.

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.

Amides↗
PubMed1985

Hydrogen exchange of individual amide protons in the F helix of cyanometmyoglobin.

Hydrogen exchange of the individual amide protons of alanine-90 (F5), glutamine-91 (F6), serine-92 (F7), and histidine-93 (F8) residues in cyanometmyoglobin of sperm whale has been studied by 1H nuclear magnetic resonance spectroscopy at 360 MHz. The amide proton resonance of F5, F6, and F7 have been assigned by use of the selective nuclear Overhauser effect between the consecutive amide protons. At pH 6.8, and in the temperature range of 5-20 degrees C, these protons show a 10(4)-fold retardation compared to the rates in free peptides. Apparent activation enthalpies for hydrogen exchange of F5, F6, and F8 protons are 18.5 +/- 0.4, 9.5 +/- 0.3, and 18.5 +/- 0.3 kcal/mol, respectively. Some implications of these results on the nature of the opening processes involved in hydrogen exchange are considered.

Amides↗
PubMed1988

Comparison of the structure and dynamics of chicken gizzard and rabbit cardiac tropomyosins: 1H NMR spectroscopy and measurement of amide hydrogen exchange rates.

The side chain and backbone mobilities of chicken gizzard tropomyosin (TM) and its nonpolymerizable derivative have been investigated by H NMR spectroscopy and amide hydrogen exchange kinetics and compared to those of rabbit cardiac TM and its nonpolymerizable derivative. Analysis of the 300-MHz H NMR spectra of native chicken gizzard and rabbit cardiac TMs and their nonpolymerizable derivatives showed that the line widths of the aromatic and histidine residues were within a factor of 2 for all four proteins, demonstrating that the side chain mobility of these residues is similar in the different TMs. Direct proton exchange-out kinetics were determined in D2O in the pD range 1.5-3.0 at 25 degrees C by H NMR spectroscopy. Multiple exponential fitting of the exchange data indicated the presence in gizzard TM of at least three kinetically distinct classes of amide hydrogens at pD 1.7 with average population sizes of 147, 74, and 61, whose rates were retarded by a factor of 10, 10(3), and 10(5), respectively, relative to the random-coil peptide poly(DL-alanine). Measurement of the direct exchange kinetics of both rabbit cardiac and nonpolymerizable gizzard TMs showed that their rate constants and population sizes were within experimental error of those for the gizzard protein, except that the fast exchanging class for cardiac TM was increased in size while that of the nonpolymerizable gizzard TM was reduced, relative to that for gizzard TM. Comparison of the exchange-out kinetics for the cardiac and gizzard proteins at pH 2.0 and 55 degrees C, where only the two slowly exchanging amide hydrogen sets are measured, again demonstrated the similarity of their kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1989

Static and transient hydrogen-bonding interactions in recombinant desulfatohirudin studied by 1H nuclear magnetic resonance measurements of amide proton exchange rates and pH-dependent chemical shifts.

With proton nuclear magnetic resonance spectroscopy at 22 degrees C and pD 4.5, individual exchange rates in the range from 2 X 10(-5) to 1 X 10(-1) min-1 were observed for 23 amide protons in recombinant desulfatohirudin. The remaining 38 backbone amide protons exchange more rapidly than 1 X 10(-1) min-1. All 23 slowly exchanging protons are located in the polypeptide segment from residue 4 to residue 42, which forms a well-defined globular domain. Three different breathing modes of this molecular region are manifested in the exchange data, which appear to be correlated with the location of the three disulfide bonds. Chemical shift changes larger than 0.15 ppm between pH 2.5 and pH 5.0 arising from through-space interactions with carboxyl groups were observed for seven backbone amide protons. Two of these shifts can be explained by hydrogen bonds in the core of the protein, Gly 25 NH-Glu 43 O epsilon and Ser 32 NH-Asp 33 O delta, and two others by intraresidual NH-O epsilon interactions in Glu 61 and Glu 62. The remaining three pH shifts for Glu 35, Cys 39, and Ile 59 imply the existence of transient interactions between the molecular core and the flexible C-terminal segment 49-65, which have so far not been characterized by nuclear Overhauser effects or other conformational constraints.

Amides↗
PubMed1990

Protein secondary structures in water from second-derivative amide I infrared spectra.

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.

Alcohol Dehydrogenase↗
PubMed2002

Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system.

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II-oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein-carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II-oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II-substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.

Amides↗
PubMed1999

Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.

Affinity Labels↗
PubMedDate not supplied

Solid phase synthesis of tertiary amines on amide REM resins: Grignard and metal hydride compatible resins.

Four new amide REM resins (AM REM 2-5) are described, and their use is illustrated for the synthesis of tertiary amines 6-9 and 13-16. Amide REM resins 4 and 5, which have a phenyl ring attached to the amide nitrogen, are found to give superior product yields and purities, and the resins are stable to a wider range of reagents and conditions compared to REM resin 1.

Amides↗
PubMedDate not supplied

A novel approach for the solid-phase synthesis of substituted cyclic guanidines, their respective bis analogues, and N-acylated guanidines from N-acylated amino acid amides.

An efficient method for the solid-phase synthesis of cyclic guanidines from N-acylated amino acid amides, bis cyclic guanidines from N-acylated dipeptides derived from orthogonally protected diamino acids, and N-acylated guanidines from disubstituted cyclic guanidines is described. The exhaustive reduction of N-acylated amino acid amides yields diamines that on treatment with cyanogen bromide lead to the formation of cyclic guanidines. Resin-bound orthogonally protected diamino acids (i.e., N(alpha)-Fmoc-N(x)-(Boc)-diamino acid, x = beta, gamma, delta, epsilon) were N-acylated following removal of the Fmoc group. Removal of the Boc functionality from the side chain then generated a primary amine. Subsequent coupling of Boc amino acids, followed by removal of the Boc group, generated dipeptides that were N-acylated. Exhaustive reduction of amide bonds of the N-acylated dipeptides generated tetraamines having four secondary amines, which upon cyclization with cyanogen bromide afforded the resin-bound trisubstituted bis cyclic guanidines. Treatment of the resin-bound disubstituted cyclic guanidines with carboxylic acids gave N-acylated guanidines. On the basis of their high yield and purity, bis cyclic guanidines derived from N(alpha)-Fmoc-N(epsilon)-Boc-lysine and N-acylated guanidines were chosen for preparation of mixture-based combinatorial libraries. Details of the preparation of these positional scanning libraries using the "libraries from libraries" concept are presented.

Amides↗
PubMedDate not supplied

Alpha-keto amide peptides: a synthetic strategy to resin-bound peptide isosteres for protease inhibitor screening on solid support.

A synthetic strategy for the formation of resin-bound internal alpha-keto amide peptides suitable for protease inhibitor screening on solid support is presented. This general approach is based on the incorporation of alpha-keto amide building blocks during solid-phase peptide synthesis (SPPS). Such dipeptidyl building blocks were accessible using the acylcyanophosphorane methodology. The acid-labile alpha-keto carbonyl functionality was protected as a 1,3-dithiolane derivative. This protective group is fully compatible with standard SPPS reaction conditions and can be efficiently removed with N-bromosuccinimide in 10% aqueous acetone. The alpha-keto amide peptides were assembled on SPOCC-1500 resin and were characterized with high-resolution magic angle spinning (HR-MAS) NMR on bead. The methodology was evaluated and tested with a variety of building blocks containing natural and nonnatural amino acid moieties.

Amides↗
PubMed2005

Amide-ligand hydrogen bonding in reverse micelles.

One approach to modeling the second coordination shell of metalloproteins is to pair amide-containing counterions with metal complexes to form hydrogen bonds in the solid state. In a more general approach, we have designed a surfactant counterion that can sustain hydrogen bonding interactions with metal complexes in solution. The surfactant is cationic and incorporates an amide as part of its headgroup to form hydrogen. The surfactant forms hydrogen bonding reverse micelles that accommodate anionic metal complexes in their polar core. In reverse micelles containing an iron(III) hexacyanide complex, spectroscopic evidence suggests that the anion is confined to the polar core region in solution. Single-crystal X-ray diffraction data on the surfactant ferricyanide system reveals a layered structure with interdigitated alkyl chains and an extensive network of hydrogen bonds that link amide groups to the cyanide ligands and to neighboring headgroups.

Amides↗
PubMed2002

Proximity effects in monolayer films: kinetic analysis of amide bond formation at the air-water interface using (1)H NMR spectroscopy.

The kinetics of amide bond formation in a monolayer film has been studied by proton NMR spectroscopy. Compression of a hexadecyl thioester of N-acetyl glycine (1) and a hexadecyl amide of glycine (2) at the air-water interface produces a single dipeptide product (4) that remains at the surface once formed. Extraction of the reaction mixture from the interface, followed by (1)H NMR spectroscopy, provides quantitative data on the rate of product formation. The kinetics of this reaction was examined as a function of surface pressure, subphase pH, and temperature. The monolayer provides an effective molarity for the reaction of approximately 500 M as compared to the bimolecular reaction of 1 and 4 in chloroform solution. The first-order rate constant for the reaction of 1 and 2 in the monolayer is less than 70-fold slower than k(cat) for condensation of the first amide bond in the enzymatic synthesis of the cyclic antibiotic gramicidin S by gramicidin S synthetase. Activation energies of the reaction were extracted from the temperature dependence of the rate constants of the reaction and are 9.9 +/- 1.0 and 2.1 +/- 0.2 kcal/mol for the chloroform solution and monolayer reactions, respectively. The pK(a) of 2 in the monolayer was estimated to be approximately 0.5 pK(a) units lower than that of related amines in solution. The lower pK(a) at the interface as compared to that in solution may be ascribed to increased electrostatic repulsion at the interface relative to solution. The rate of reaction in the monolayer was also followed by monitoring changes in surface area as a function of time. The rate constant for the reaction of 1 and 4 as determined by changes in surface area differs significantly from the rate determined by NMR. The results indicate that measurements of surface area versus time may yield erroneous rate constants for reactions in monolayers.

Air↗
PubMed2002

A general and efficient copper catalyst for the amidation of aryl halides.

An experimentally simple and inexpensive catalyst system was developed for the amidation of aryl halides by using 0.2-10 mol % of CuI, 5-20 mol % of a 1,2-diamine ligand, and K(3)PO(4), K(2)CO(3), or Cs(2)CO(3) as base. Catalyst systems based on N,N'-dimethylethylenediamine or trans-N,N'-dimethyl-1,2-cyclohexanediamine were found to be the most active even though several other 1,2-diamine ligands could be used in the easiest cases. Aryl iodides, bromides, and in some cases even aryl chlorides can be efficiently amidated. A variety of functional groups are tolerated in the reaction, including many that are not compatible with Pd-catalyzed amidation or amination methodology.

Amides↗
PubMed2003

Detection of amide I signals of interfacial proteins in situ using SFG.

In this Communication, we demonstrate the novel observation that it is feasible to collect amide signals from polymer/protein solution interfaces in situ using sum frequency generation (SFG) vibrational spectroscopy. Such SFG amide signals allow for acquisition of more detailed molecular level information of entire interfacial protein structures. Proteins investigated include bovine serum albumin, mussel protein mefp-2, factor XIIa, and ubiquitin. Our studies indicate that different proteins generate different SFG amide signals at the polystyrene/protein solution interface, showing that they have different interfacial coverage, secondary structure, or orientation.

Absorption↗
PubMed2005

A quantitative reconstruction of the amide I contour in the IR spectra of globular proteins: from structure to spectrum.

The Amide I contours of six globular proteins of varied secondary structure content along with a peptide model for collagen and pulmonary surfactant protein C have been simulated very closely by using a modified GF matrix method. The starting point for the method uses the three-dimensional structure as obtained from the Protein Data Bank. Elements of the interactions between peptide groups (e.g., transition dipole coupling) are very sensitive to tertiary structure, thus the current formalism demonstrates that the Amide I contour may be useful for a more detailed probe of 3-D conformation that goes beyond the traditional use of this band to probe the percentages of particular elements of secondary structure. For example, postulated changes to a known structure can be tested by comparing the new simulated band to the experimental band. A number of refinements to the transition dipole interaction calculation have been made. Most of the important interactions between the C=O oscillators that define the Amide I mode appear to have been identified, including through space transition dipole coupling, through valence bond and through hydrogen bond coupling. The eigenvector matrix produced by the method permits the contribution of each peptide group to the spectrum to be precisely determined. Analysis of the results shows that the often-used structure-frequency correlations are at best approximate and at worst misleading. The subbands from helices, sheets, turns, and loops are much broader and more overlapped than has been commonly assumed. Furthermore, the traditional alpha-helical marker band may be substantially distorted in short segments. Difference spectra based on isotope editing, a technique thought capable of revealing the spectral contributions of individual peptide groups, are shown to be prone to misinterpretation.

Amides↗