Amino acids, labeling

Abstract. A smooth empirical potential is constructed for use in off-lattice protein folding studies. Our potential is a function of the amino acid labels and of the distances between the Ca atoms of a protein. The potential is a sum of smooth surface potential terms that model solvent interactions and of pair potentials that are functions of a distance, with a smooth cutoff at 12 Angstrom. Techniques include the use of a fully automatic and reliable estimator for smooth densities, of cluster analysis to group together amino acid pairs with similar distance distributions, and of quadratic progrmnming to find appropriate weights with which the various terms enter the total potential. For nine small test proteins, the new potential has local minima within 1.3-4.7A of the PDB geometry, with one exception that has an error of S.SA. 

The estimated correlation times for the loop domains of the order of 10 4 s are obtained for the suppressed peaks in the [l-13C]amino-acid-labelled bR, including Gly, Ala, and Leu residues as shown in Figure 24C. The loop dynamics can be also examined by measurements of the 13C-1H dipolar couplings by DIPSHIFT experiment in which fluctuations of the Co,-Cp vector result in additional motional averaging as order parameters, in addition to the rotation of Ala methyl groups which scales the dipolar... 

Amino acids labeled with DNS-C1 were determined using the Ru(bpy)32+ CL reaction after HPLC separation with a reversed-phase column [104, 105], DNS derivatives are expected to produce intense CL owing to their secondary and tertiary amino groups. The detection limit for DNS-Glu was 0.1 pM (2 pmol/ injection). Although underivatized amino acids could be detected by Ru(bpy)32+ CL, the DNS derivatives showed improved detection limits by three orders of magnitude [105], An approach to convert primary amines to tertiary amines was also reported [106], In this method, divinyl sulfone (DVS) was used for a cycloaddition reaction of primary amines (Fig. 19). The DVS derivatives after HPLC separation were sensitively detected (e.g., detection limits for propylamine and 3-aminopentane were 30 and 1 pmol, respectively). 

Gruetter, R., Novotny, E. J., Boulware, S. D. etal. Localized 13C NMR spectroscopy of amino acid labeling from [ 1—13C] D-glucose in the human brain. /. Neurochem. 63 1377-1385, 1994. 

Protein Perdeuteration and Selective Amino Acid Labeling... 

Protein Perdeuteration and Selective Amino Acid Labeling 464 NMR-Based Drug Design Techniques 465... 

There is a continuing demand for amino acids labeled with deuterium, or N, in particular for multidimensional NMR applications, and the broad specificity of LeuDHs has led to its use in several procedures for... 

P. Damhaut, C. Lemaire, A. Plevenaux, C. Brihaye, L. Christiaans, D. Comar, No-carrier-added asymmetric synthesis of alpha-methyl-alpha-amino acids labeled with fluorine-18, Tetrahedron 53 (1997) 5785-5796. 

Name of dye available Amino acids labeled Sensitivity ... 

A more extensive study of mobilities of 3H- and 14C-labeled amino acids again found that amino acids labeled with 14C at Cl or C2 are retained on the column, relative to the unlabeled forms.135 Lysine is an exception. Tritiation at C3 also increases the retention time, but tritiation at C2 of glycine or at C4, C5, or C6 of lysine decreases it, and large decreases are seen with methionine tritium-labeled in the methyl and with tyrosine tritium-labeled at C3, 5. The 14C IEs can be attributed to a decrease of acidity, but the IEs of distant 3H may be due to hydrophobic interactions with the resin. A remarkable result is that intramolecular isotopic isomers (isotopomers) can be distinguished on the basis of their chromatographic mobilities. 

An optically gated injection was demonstrated for the CZE separation of four amino acids labeled with 4-chloro-7-nitrobenzofurazan (NBD-F) in a one-channel chip [576] or a four-channel chip [577]. The gating beam was used to continuously photobleach the sample, except for a short time during injection by interrupting the beam (100-600 ms) using an electronic shutter. With only a sample reservoir and a waste reservoir, the sample continuously flowed electrokinetically. Six consecutive separations of the same sample mixture have been accomplished in under 30 s [576,577],... 

In another method the nitrogen of the N-terminal amino acid is reacted with dansyl chloride. The resulting sulfonamide bond is quite resistant to hydrolysis, so the N-terminal amino acid, labeled with the dansyl group, is readily determined after the peptide bonds have been hydrolyzed. 

Besides amino acids, labeled saa harides are suitable for the labeling of glycoproteins. In this manner, glycoproteins have been labeled with H-mannose H-glucosamine N-acetylmanosamine Saccharides may also be allowed to react with proteins after periodate oxidation... 

Figure 1. (a) View of the inside of the Methanol Dehydrogenase (MDH) enzyme with the active site in stick model. The solid surface represents the solvent-accessible MDH external surface showing the binding pocket, (b) View from the binding pocket of the entire MDH active site. Amino acids labels denote their location in the sequence obtained from the entry 1W6S (Methylobacterium Extorquens W3A1 ) of the Protein Data Bank. 

Gliotoxiii.—In contrast to the recent report that the cyclic dipeptide (121) is not incorporated into gliotoxin (123) in Penicillium terlikowskii, it has recently been found that this cyclic dipeptide, with both constituent amino-acids labelled, is efficiently incorporated into gliotoxin (123) in Trichoderma viride and without alteration in isotope ratio. Thus, (123), like brevianamide A and echinulin, originates from a cyclic dipeptide. The difference in the two results could be due to the different organisms used in the two experiments, but is was suggested that the probable reason was associated with the large excess of (121) used in the first experiment. 

However, recent advances in trypsin cleavage and subsequent mass spectrometric identification of peptide fragments, coupled with the development of photoactive derivatives of propafenones, have enabled Chiba and colleagues to identify specific amino acids labeled during cross-linking [197]. Quantitative analysis of photolabeling indicates major sites for reactivity within TM3 (methionine 197), TMS (methionine 311), TMS (methionine 769), and TMll (methionine 951) (see Table 1.1 and Ref. [197]). Minor peaks were identified within several other TM a-helices including TMl and 12 [197] (Table 1.2). 

A compromise between specific and uniform labelling of proteins is selective labelling, where certain amino acids or parts of amino acids are labelled. Bacteria fed with isotope-labelled versions of the amino acids Arg, Cys, His, lie. Leu, Lys, Met, Phe, Pro, Trp, Tyr and Val will produce proteins with these amino acids labelled due to... 

Although this approach works in principle with uniformly 15N-labeled protein, it is more favorable in combination with selective amino acid labeling. First, in the absence of a spin-labeled inhibitor, the specific amino acid resonances are identified when compared to the spectra recorded from uniformly labeled kinases. The number of peaks is reduced and therefore more manageable if the spin-labeled inhibitor is added in a second step. Especially in regions with aggravated signal overlap, the selective labeling technique clearly separates the peaks and, therefore, enables the quantification of the induced peak attenuation. 

A peptide from pyruvate kinase labeled with oADP (55) and one from ferre-doxin-NADP reductase labeled with oNADP (75) have been isolated and characterized. These are exceptions. Despite the large number of papers describing the kinetics of affinity labeling by periodate-oxidized nucleotides, there are very few reports of the identification of particular amino acids labeled by these reagents within determined peptide sequences. For enzyme products other than a Schiff base reducible by NaBHU, the instability of the products in the proteolytic digests of modified enzymes under conditions of peptide purification has precluded isolation of labeled peptides in most cases. 

Yields (%) are calculated based on input N2 or CO2 (for HCHO) from 100 mm CO2, 100 mm N2 and 100 mL H2O with or without 2 mmoles CaCCb in a 3.1 L flask at 23 C. Also included is an experiment with 10 mm of O2 added to the gas mixture. The results with a reducing atmosphere (% yields based on starting carbon in the form of methane) are given for comparison [taken from (3, 4). Yields of THAA (Total Hydrolyzable Amino Acids labeled in plate 2) are shown after hydrolysis in the absence (-) or presence (+) of ascorbate. The controls are not sparked and represent blanks. ND = not determined. 

Adda) in position 5 followed by D-glutamic acid (D-Glu) y-linked to N-methyl-dehydroalanine (Mdha) (50-52). The main stmctural variations of the more than 50 variants isolated so far, are related to mutations at the two L-amino acids labeled with X and Y in Fig. 4 (53) correspondingly, microcystin variants are named according to the residues X and Y (54). Among all the variants identified so far the most abundant and, correspondingly, the most studied is the microcystin-LR. 

The H CN (or CN, if the reaction is done under basic conditions) synthon has been mainly used to extend the carbon chain by one carbon. For example, cyanide ion has been used in the synthesis of amino acids labelled in the carboxylate group. This is accomplished using the high pressure-high temperature modification of the Bucherer-Strecker synthesis. In this reaction, bisulphite addition complex of an aldehyde reacts with cyanide ion in the presence of ammonium carbonate to form a hydantoin, which is then converted into the amino acid by basic hydrolysis (equation 61). 

Touring the past decade a number of research laboratories have devel-oped procedures for the rapid synthesis of amino acids labeled with short-lived, positron-emitting radionuclides. These tracers make possible the study of regional amino-acid metabolism in the living organism by external detection of the y photons produced by positron annihilation. This decay mode also permits the determination of label distribution in transverse sections through the body by means of positron emission... 

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