[Synthetic analogues of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi].

Analogues of latarcins Ltc1 and Ltc3b, antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi capable of formation of amphiphilic structures in membranes without involvement of disulfide bonds, were synthesized. The amino acid sequences of the analogues correspond to immature forms of these peptides, each of them containing an additional C-terminal amino acid residue. It is concluded from the study of the biological activity of the synthesized peptides that the posttranslational C-terminal amidation of Ltc3b is a functionally important modification that ensures a high activity of the mature peptide. The lipid composition was shown to affect the interaction of synthesized peptides with artificial membranes. The analogue of Ltc3b manifested the highest activity on cholesterol-containing membranes. The mechanism of action of the studied antimicrobial peptides on membranes is discussed.

Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje.

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

[Interaction of topotecan, DNA topoisomerase I inhibitor, with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. IV. Topotekan sviazyvaetsia preimushchestvenno s GC-parami osnovanii DNK.

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.

[Interaction of topotecan--a DNA topoisomerase inhibitor--with dual-stranded polydeoxyribonucleotides. I. Dimerization of topotecan in solution]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. I. Dimerizatsii topotekana v rastvore.

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 +/- 0.7) x 10(3) M-1.

Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense.

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn2+, Mg2+, and Co2+ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn2+-activated and Mg2+-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form alpha-helices and 13% beta-strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

[Synthesis isomeric 4-prolylamines and 4,4'-diprolylamines]. / Sintez izomernykh 4-prolinilaminov i 4,4'-diprolinilaminov.

Starting from the previously described tert-butyl esters of 4-epimeric N-benzyloxycarbonyl-4-hydroxyprolines and N-benzyloxycarbonyl-4-trans- and 4-cis-trifluoroacetamidoproline tert-butyl esters, the corresponding un-protected 4-aminoprolines and a number of their partially protected derivatives were synthesized via the intermediate 4-O-mesyl and 4-azide derivatives. The reductive amination of N-benzyloxycarbonyl-4-oxoproline tert-butyl ester with ammonium acetate led to N-benzyloxycarbonyl-4-cis-4'-cis- and 4-cis-4'-trans-diprolinylamines. The 1H NMR and CD spectra of the synthesized compounds are described.

Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Confocal spectral imaging analysis of intracellular interactions of mitoxantrone at different phases of the cell cycle.

It is suggested that the cytotoxicity of anticancer agent mitoxantrone (MITOX) is related to a complex combination of molecular interactions which lead to slowing of S phase traverse and arresting of cells in G2 phase of the cell cycle or even to an apoptosis at high concentration of MITOX. Here intracellular molecular interactions of MITOX were visualised and studied using the confocal spectral imaging technique in synchronised K562 cells. Localisation, quantitative distributions of MITOX in the polar environment, MITOX bound to hydrophobic cellular structures (MITOXphob), nucleic acid-related complexes of MITOX (MITOXNA) and relative distributions of naphthoquinoxaline (NQX) metabolite and intrinsic cellular fluorescence of porphyrins were measured within cytoplasmic and nuclear compartments (chromosomes) of the G2, S, and M cells treated with 10 or 2 microM of MITOX for 1 hour. Colocalisation of MITOX, NQX metabolite and sites of intrinsic cellular fluorescence indicates an accumulation of MITOX within or near mitochondria. One may suppose that due to high concentration MITOX can compete with natural substrates for binding to the enzymes thus affecting the normal functioning of a mitochondria. A remarkable redistribution of MITOX and its complexes occurs in the M cells. In particular, a prominent amount of MITOX is associated with the surface of chromatids but not with the cytoplasmic structures in M cells. At the present time the exact location of the sites of MITOX accumulation in the M cells is not known. It is thought to be some cytoskeleton/microtubule structures associated directly with the chromosomes. Selective labelling of particular cytoskeleton structures and/or proteins in MITOX treated cells is in the progress now and the question will be addressed using the CSI technique.

[Study of localization and molecular interactions of biologically active compounds in living cells and tissue slices based on confocal microspectroscopy and reconstruction of the spectral images]. / Issledovaniia lokalizatsii i molekuliarnykh vzaimodeistvii biologicheski aktivnykh soedinenii v zhivykh kletkakh i srezakh tkanei na osnove metoda konfokal'noi mikrospektroskopii i rekonstruktsii spektral'nykh izobrazhenii.

The confocal spectral imaging (CSI) technique is described, its basic principles are considered, and a brief review of its applications to the study of biologically active compounds (BAC) within living cells and in tissue slices is presented. This technique is based on measurements and analysis of fluorescence or resonance Raman spectra in each point of the specimen under microscope with a three-dimensional resolution of about cubic micrometer. This technique is applicable to the study of stained fluorescent and nonfluorescent compounds. Unlike the conventional approaches based on the optical microscopy, the CSI technique opens the opportunity for the identification of complexes and microenvironment of BAC in intact cells and thin tissue slices (slices or sections), as well as for the analysis of localization and distribution of compounds of interest and their complexes in cellular organelles and tissue structures. The use of CSI technique in combination with the conventional biochemical and cytological methods makes it possible to significantly expand the informativeness of investigation of modes of action of new BAC.

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

Spectroscopic and biochemical characterisation of self-aggregates formed by antitumor drugs of the camptothecin family: their possible role in the unique mode of drug action.

We describe the effect strongly influencing the biological activity of some camptothecin (CPT) drugs, the inhibitors of DNA topoisomerase I (topo I), namely, the formation of J-type aggregates in an aqueous buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20-S-camptothecin (20(S)CPT), 10,11-methylenedioxy-CPT (10,11-CPT) and 7-ethyl-10-hydroxy-CPT (SN38). The aggregates were found to be stereospecific, not being detectable for the 20(R)-stereoisomer of CPT. They were formed by the stacking interaction between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. The aggregates were stable at acidic and neutral pHs, but dissociated at basic pHs. Self-aggregation prevented hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. Addition of BSA did not induce either disaggregation or hydrolysis of the lactone ring, whereas the monomeric form of the drugs was shown to undergo rapid conversion to an inactive carboxylate form in the presence of human serum albumin [5]. The drugs did not form the aggregates in the presence of topo I. Moreover, rapid dissociation of the aggregates was observed if a self-aggregated drug solution was added to topo I alone or to the DNA-topo I cleavage assay. Neither DNA alone nor oligonucleotides derived from the sequences of the CPT-enhanced or topo I-induced cleavage sites in SV40 plasmid DNA induces changes in the aggregation state of the drugs. These observations are indicative of interaction between the aggregates and topo I. The aggregates were found to penetrate within the cells with much higher efficiency than a monomeric form of the drugs. Cellular uptake of aggregated and nonaggregated species correlated well with cytotoxic effects produced by the drug. In this manner, CPT's self-aggregation should be regarded as a favourable phenomenon producing species with a more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.

Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis.

Studying mechanisms of drug antitumor action is complicated by the lack of noninvasive methods enabling direct monitoring of the state and interactions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming this problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, 8-bis-[([2-(2-hydroxyethyl)-amino]ethyl)amino]-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spectra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target complexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affecting drug fluorescence. MITOX exposed to aqueous intracellular environment, MITOX bound to hydrophobic cellular structures, complexes of MITOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These states and complexes are known to be immediately related to the antitumor action of the drug. The results obtained present a basis for the subsequent quantitative analysis of concentration and time-dependent accumulation of free and bound MITOX within different compartments of living cancer cells.

Quantitative confocal spectral imaging analysis of mitoxantrone within living K562 cells: intracellular accumulation and distribution of monomers, aggregates, naphtoquinoxaline metabolite, and drug-target complexes.

Confocal spectral imaging (CSI) technique was used for quantitative analysis of the uptake, subcellular localization, and characteristics of localized binding and retention of anticancer agent mitoxantrone (MITOX) within human K562 erythroleukemia cells. The CSI technique enables identification of the state and interactions of the drug within the living cells. Utilizing this unique property of the method, intracellular distributions were examined for monomeric MITOX in polar environment, MITOX bound with hydrophobic cellular structures, naphthoquinoxaline metabolite, and nucleic acid-related complexes of MITOX. The features revealed were compared for the cells treated with 2 microM or 10 microM of MITOX for 1 h and correlated to the known data on antitumor action of the drug. MITOX was found to exhibit high tendency to self-aggregation within intracellular media. The aggregates are concluded to be a determinant of long-term intracellular retention of the drug and a source of persistent intracellular binding of MITOX. Considerable penetration of MITOX in the hydrophobic cytoskeleton structures as well as growing accumulation of MITOX bound to nucleic acids within the nucleus were found to occur in the cells treated with a high concentration of the drug. These effects may be among the factors stimulating and/or accompanying high-dose mitoxantrone-induced programmed cell death or apoptosis.

Interactions of lactone, carboxylate and self-aggregated forms of camptothecin with human and bovine serum albumins.

Pronounced differences in the interactions of monomeric (lactone and carboxylate) and the J-type self-aggregated form of camptothecin (CPT), an inhibitor of DNA topoisomerase (topo) I, with human (HSA) and bovine (BSA) serum albumins were observed by using circular dichroism (CD) spectroscopy. HSA binding changes the geometry of the covalent structure of CPT due to hydrophobic contacts of the chromophore within the protein interior. The carbonyl group of the ring D of CPT (Fig. 1A) interacts with the positively charged amino acid residues of HSA. Interaction with HSA induces disaggregation of the J-type self-aggregates of CPT. On the other hand, neither heat-denatured HSA nor native BSA participated in binding of the lactone or carboxylate or self-aggregate forms of CPT. Analysis of HSA and BSA homology within the IIA and IIIA principle ligand-binding structural domains suggests that the binding site for the CPT chromophore is located in subdomain IIA. Hydrophobic contacts with Leu-203, Phe-211, and Ala-215 and electrostatic interactions with Lys-199 and/or Arg-222 of HSA may play a key role in formation of the drug-HSA complex.

[Novel reagent for labeling of biomolecules--activated derivative of pyrene dichromophore with excimeric fluorescence]. / Novyi reagent dlia mecheniia biomolekul--aktivirovannoe proizvodnoe pirenovogo bikhromofora s éksimernoi fluorestsentsiei.

N-[2-(1-pyrenyl)ethyl]-1-pyrenylacetamide, bis[2-(1-pyrenyl)ethyl]amine, and N,N-bis[2-(1-pyrenyl)ethyl]succinamide were synthesized from 1-pyrenylacetic acid. These compounds contain adjacent pyrene residues and display excimer fluorescence. The latter compound, as a pentafluorophenyl ester, was used to prepare fluorescently labeled oligodeoxyribonucleotide (5)CAGGAAACAGCTATGAC. For N,N-bis[2-(1-pyrenyl)ethyl]succinamide, the excimer-to-monomer fluorescence ratio and intensity of fluorescence in water-methanol solutions changed in the presence of single-stranded and double-stranded oligonucleotides, upon attachment to an oligonucleotide, and upon hybridization of the resulting conjugate with the complementary nucleotide sequence.

Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.

[Physico-chemical properties of DNA-dependent RNA-polymerase from Escherichia coli and its subunits]. / Fiziko-khimicheskoe izuchenie DNK-zavisimoi RNK-polimerazy iz Escherichia coli i ee otdel'nykh sub''edinits.

CD and UV spectroscopy were employed to study at different temperatures the conformational states of the DNA-dependent RNA polymerase core- and holo-enzymes, as well as of its alpha and beta subunits. Both core- and holo-enzyme were shown to have a higher percentage of regular structures than the separate subunits. CD and fluorescence methods were used to monitor the complex formation between rifamycin SV or its derivative, rifampicin, with the RNA polymerase from the E. coli wild and mutant (Rpo B255) types, the former enzyme being sensitive and the latter being resistant to these antibiotics. Complexation led to concomitant changes in the conformation of antibiotics and local structural rearrangements of the protein in vicinity of the binding site which comprises at least one tryptophan residue in a hydrophobic microenvironment.