Analysis of tryptophan surface accessibility in proteins by MALDI-TOF mass spectrometry

Surface accessible amino acids can play an important role in proteins. They can participate in enzyme's active center structure or in specific intermolecular interactions. Thus, the information about selected amino acids' surface accessibility can contribute to the understanding of protein structure and function. In this paper, we present a simple method for surface accessibility mapping of tryptophan side chains by their chemical modification and identification by MALDI-TOF mass spectrometry. The reaction with 2-hydroxy-5-nitrobenzyl bromide, a common and highly specific covalent modification of tryptophan, seems to be very useful for this purpose. The method was tested on four model proteins with known spatial structure. In the native proteins (1) only surface accessible tryptophan side chains were found to react with the modification agent and (2) no buried one was found to react at lower reagent concentrations. These results indicate that the described method can be a potent tool for identification of surface-located tryptophan side chain in a protein of unknown conformation.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification.

A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast. As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used. The residue which is modified by the procedure described has been identified as Trp-193. There are either one or two molecules of the modifying agent being added to this tryptophan side chain. The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed. However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification. It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme. Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.     

Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?

The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

Chemical modification of the tryptophan residue in toxin B from the venom of the Indian cobra

The single tryptophan residue in toxin B has been converted into N′-formylkynurenine by ozonization in anhydrous formic acid, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (HNB) bromide and 2-nitro-4-carboxyphenylsulphenyl (NCPS) chloride. Amino acid analyses of such modified derivatives show these reactions to be specific for tryptophan without significant effect to other amino acids. Ozonized toxin B has a residual toxicity of 80 %, and other tryptophan modified toxins retain at least half the toxicity of native toxin B. Each modified derivative gave a single fused precipitin line with native toxin on immunodiffusion against antitoxin B sera. In heterologous precipitin reactions, no significant decreases in antigenic activity of the modified derivatives were observed. The tryptophan residue at position 25 may, therefore, be part of neither the active site nor the antigenic site.     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

Antithrombin conformation and the catalytic role of heparin. II. Is the heparin-induced conformational change in antithrombin required for rapid inactivation of thrombin?

The role of antithrombin conformation in heparin-catalyzed inhibition of thrombin was investigated using antithrombins modified with the tryptophan reagent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB). Affinity fractionation of HNB-labeled antithrombin (0.6-0.7 mol of HNB/mol of protein) on heparin-Sepharose using a linear salt gradient allowed separation of three singly labeled protein species and a fourth HNB-antithrombin species which co-eluted with unlabeled protein. Conformational alterations induced by heparin binding to each of the labeled antithrombins were assessed by spectroscopic techniques, including protein fluorescence, difference spectroscopy in the ultraviolet-visible range, and circular dichroism. Comparison of spectra of the labeled proteins in the presence and absence of added heparin indicated changes to occur in protein conformation at the sites of the bound HNB moieties and at aromatic amino acid residues within the protein matrix. These spectroscopic alterations mimicked changes induced by heparin in the native protein, but were reduced in magnitude. Rates of thrombin inactivation by the labeled antithrombins were measured over a wide range in both heparin concentration and inhibitor concentration to determine maximal rates of protease inactivation. The kinetic analysis indicated that each of these HNB-antithrombin derivatives, which undergo the heparin-induced changes to varying extents, can react with thrombin at the same maximal rate. Thus, this series of chemically modified antithrombin species demonstrated that the conformational change which is induced in antithrombin by heparin does not render the protein intrinsically more reactive toward thrombin.     

Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

Modification of tryptophan residues in immunoglobulin M by 2-hydroxy-5-nitrobenzyl bromide

The modification of tryptophan residues in monoclonal immunoglobulin M (IgM) by 2-hydroxy-5-nitrobenzyl bromide (RK) was studied at pH 2.0-2.85 and 7.0 and a RK to tryptophan molar ratio (K) from 1 to 40. At pH 2.85, the number of RK residues bound to IgM (N) in account to one HL-fragments does not exceed 10 (the HL-fragment of IgM contains 14 tryptophan residues); the plot of N vs K reaches a plateau at K greater than 20. When the pH is lowered to approximately 2, N rises to approximately 15, but the plateau is not reached. At pH 7.0, the modified IgM with N greater than 1 gives a sediment, while the product with N approximately equal to 1 remains in solution. Evidently, the latter contains the most accessible tryptophan residue (calculated per one HL-fragment). This residue was found to be one of the three residues localized in the C mu 2-domain and the adjoining N-terminal part. The possibility of multiple modification of tryptophan residues during the RK interaction with IgM in acid medium at high values of K is discussed.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

Nitrated and oxidized products of a single tryptophan residue in human Cu,Zn-superoxide dismutase treated with either peroxynitrite-carbon dioxide or myeloperoxidase-hydrogen peroxide-nitrite

We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.     

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.     

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.     

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.     

A newly synthesized,potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.     

Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV concomitant with tryptophan alkylation

Reaction of Cerebratulus lacteus toxin B-IV with 2-hydroxy-5-nitrobenzyl bromide at pH 4.5 results in modification of toxin tryptophan residues and loss of biological activity. With relatively small reagent excesses, one tryptophan per molecule is modified without major effect on toxicity. Further reaction results in modification of a second residue of tryptophan and loss of at least 95% of the toxic activity. Modification of one or both tryptophan residues is without significant effect on the secondary structure of the protein. The specificity of each phase of the reaction has been assessed by fingerprint analysis of peptides derived from toxin modified to differing extents with 2-hydroxy-5-nitrobenzyl bromide. It is thus possible to show that tryptophan-5 reacts first and tryptophan-30 only under more rigorous conditions. It thus appears that tryptophan-30 is essential for full neurotoxic activity.     

Accessibility of tryptophan residues in immunoglobulin M molecule as an indicator of its conformational variability

The accessibility of tryptophan residues in immunoglobulin M to modification with the Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) was used as an indicator of its conformational variability. Of 14 tryptophan residues (per HL-fragment) in the native IgM, only one (presumably Trp312 in the mu-chain) was the most accessible. Irreversible acid- or temperature-induced conformational changes of IgM increased almost 2-fold the number of accessible tryptophan residues. After partial enzymatic deglycosylation of IgM (especially by an intense splitting of mannose), all tryptophan residues became inaccessible. Modification of the most accessible tryptophan residue increased 2- to 3-fold the number of tyrosine residues accessible to nitration with tetranitromethane. Using the spin label method, it was demonstrated that modification of four tryptophan residues in IgM considerably decreased the mobility of the Cmu 3 domain together with an essential drop in. the solubility of the modified IgM.