Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana

The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.     

Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.     

The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties

Special analysis of the tryptophan residue localization in the structure of the macromolecule of Pseudomonas aeruginosa azurin made it possible to prove many explanations in the existing literature of the extraordinary fluorescence properties of this protein, to choose between various contradictory conclusions and in some cases even to make new interpretations of the known experimental data. It has been revealed that the microenvironment of the tryptophan residue is in principle formed by non-polar hydrocarbon groups. The density of the microenvironment is not very high and there are cavities around the ring. The conformation of the side chain of the tryptophan residue is unstrained. These results have been analysed in connection with available data on the unique short-wave fluorescence spectrum position and the existence of the high-frequency indole ring mobility with significant amplitude. Judging by the distance between tryptophan and tyrosine residues and their mutual orientation, the conclusion was made that there is no energy transfer from Tyr 72 to tryptophan and that the efficiency of the energy transfer from Tyr 108 to tryptophan is about 0.5. The mechanism of the dramatic increase in fluorescence efficiency when the copper atom is removed has been discussed with due regard to the fact that the 'blue' copper centre is displaced from the indole ring by more than 10 A.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

A possible biological role of the electron transfer between tyrosine and tryptophan. Gating of ion channels.

Experiments have demonstrated that four tryptophan residues are located near the tetrodotoxin binding site in Na+ channels, and that conserved tyrosine and tryptophan residues are located in the pore-forming region of voltage-sensitive K+ channels. This paper proposes an activation mechanism involving electron transfer between these residues. The K+ channel may be closed by four tyrosine residues forming hydrogen bonds with each other. After electron transfer, these hydrogen bonds will be broken, thereby opening the channel. The Na+ channel could be activated by a similar mechanism. This idea can be tested directly by observing tyrosine or tryptophan radicals when the channels are in the open state.     

Evidence from pulse radiolysis and flash photolysis of the environment of tryptophan and tyrosine in the coat protein of fd phage.

Charge transfer has been observed between oxidised tryptophan-26 units and the tyrosine-21 or −24 of the coat protein of fd phage. The transfer is likely to be intermolecular. The rates suggest that the aromatic units are in a rigid region and that they may have at least two different environments. No apparent interaction occurs with the DNA, consistent with tryptophan and tyrosine units not being in contact with the bases.     

Evidence from pulse radiolysis and flash photolysis of the environment of tryptophan and tyrosine in the coat protein of fd phage

Charge transfer has been observed between oxidised tryptophan-26 units and the tyrosine-21 or -24 of the coat protein of fd phage. The transfer is likely to be intramolecular. The rates suggest that the aromatic units are in a rigid region and that they may have at least two different environments. No apparent interaction occurs with the DNA, consistent with tryptophan and tyrosine units not being in contact with the bases.     

Triplet state energy transfer in several proteins.

Energy transfer between excited triplet states of aromatic amino acid residues was observed at 1.4 degrees K. The distance necessary for energy transfer between monomeric tyrosine and tryptophan residues was determined to be roughly 63 A. Total phosphorescence decay rate constants for several proteins were determined while emission corresponding to tyrosine and tryptophan residues was monitored. The observed decay rate constants are interpreted in terms of intramolecular interactions of the polypeptide residues.     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

Intrinsic UV-fluorescence of lysozyme and microenvironment of its tryptophan residues]

The localization of tryptophan residues in hen egg-white lysozyme macromolecule was studied on the basis of the known 3D structure. The polarity and packing density of their microenvironments were evaluated. All residues that can affect the tryptophan fluorescence were revealed. It was shown that the orientation of these active groups relative to the indole ring of tryptophan plays a dramatic role in the efficiency of their influence. Tryptophan--tryptophan nonradiative energy transfer was evaluated from distances between tryptophan residues and their mutual orientation. The conformation of the side chains of tryptophan residues was determined. Special attention was paid to microenvironment of Trp108 responsible for the minor absorption band at 305 nm.     

Synthesis and DNA binding properties of tryptophan linked monocationic netropsin analogues

One tryptophan (Trp-Net) and two tryptophan (Trp-Trp-Net) residues have been linked to the amino terminus of a minor groove binding Netropsin analogue. DNA metling measurements indicate that Trp-Trp-Net binds significantly stronger than Trp-Net to double helical DNA. Both compounds induce helix extension as measured by changes in DNA viscosity indicating the possibility of intercalation of the tryptophan indole ring.     

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.     

Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains. Assignments and structural interpretation.

Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion. To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains. Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer [fd(Wd5)], (b) ten deuterio-alanines per protomer [fd(10Ad3)], and (c) both deuterio-tryptophan and deuterio-alanine [fd(Wd5 + 10Ad3)]. The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988). Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data. The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan [fd(Wd5) and fd(Wd5 + 10Ad3)], show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor. (b) The planar indole ring exists in a hydrophilic environment. (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees. With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum. The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Identification of Tyr413 as an active site residue in the flavoprotein tryptophan 2-monooxygenase and analysis of its contribution to catalysis

The flavoenzyme tryptophan 2-monooxygenase catalyzes the oxidation of tryptophan to indoleacetamide, carbon dioxide, and water. The enzyme is a homologue of l-amino acid oxidase. In the structure of l-amino acid oxidase complexed with aminobenzoate, Tyr372 hydrogen bonds with the carboxylate of the inhibitor in the active site. All 10 conserved tyrosine residues in tryptophan 2-monooxygenase were mutated to phenylalanine; steady state kinetic characterization of the purified proteins identified Tyr413 as the residue homologous to Tyr372 of l-amino acid oxidase. Y413F and Y413A tryptophan 2-monooxygenase were characterized more completely with tryptophan as the substrate to probe the contribution of this residue to catalysis. Mutation of Tyr413 to phenylalanine results in a decrease in the value of the first-order rate constant for reduction of 35-fold and a decrease in the rate constant for oxidation of 11-fold. Mutation to alanine decreases the rate constant for reduction by 200-fold and that for oxidation by 33-fold. Both mutations increase the K(d) value for tryptophan and the K(i) values for the competitive inhibitors indoleacetamide and indole pyruvate by 5-10-fold. Both mutations convert the enzyme to an oxidase, in that the products of the catalytic reactions of both are indolepyruvate and hydrogen peroxide. The V/K(trp)-pH profiles for the Tyr413 mutant enzymes no longer show the pK(a) value of 9.9 seen in that for the wild-type enzyme, allowing identification of Tyr413 as the active site residue in the wild-type enzyme which must be protonated for catalysis. Substitution of Tyr413 abolishes the formation of the long wavelength charge transfer species observed in the wild-type enzyme. The data are consistent with the main role of Tyr413 being to maintain the correct orientation of tryptophan for effective hydride transfer and imino acid decarboxylation.     

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.     

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.     

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.