SynopsisThe activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-Zethylhexylsulfosuccinate (AOT)/isooctane/H,O, cetyltrioctylammoniumbromide (CTAB)/CHCl,, isooctane/H,O tetraethyleneglycoldodecylether (E0,C ,,)/isooctane/H,O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the E04C12 or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. BWZ. Chern. 256, 837-8431 dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG3, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG3, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w,, using the chromophoric substrate 3,4dinitrophenyl-tetra-N-acetyl-0-D-chitotetraoside (3,4-DNP-NAG4 ). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k,, tends to be smaller than in aqueous solution (with quite similar KM), whereas in the EO,C,, system (at pH 7.0) the turnover number is larger and K M is smaller than in water. In the AOT system, the kinetic parameters a t pH 7.0 are also quite comparable to those found in water.
SynopsisIn chloroform solution, the D,L-alteI'nating stereo-co-oligopeptide HCO-L-Phe-(DPhe-L-Phe),-OMe (I) forms three major species, two of which are dimeric and one tetrameric. One of the two dimeric species gives a specific set of 'H-nmr signals at 25°C; the other, together with the tetrameric species, gives another set of resonance signals. In a carbon tetrachloride or cyclohexane solution at 25"C, I forms virtually only the tetrameric species. From the nmr data, it can be shown that the dimeric and tetrameric species, that are in rapid equilibrium with each other in chloroform solutions, are a right-handed t tP5 helical dimer and the head-to-head (formylends-to-formylends) dimerization product of this dimer. It is suggested that the linear gramicidins may also form head-tohead dimers of parallel P helices, as observed for the model oligopeptide I.
SynopsisThe circular dichroism (CD) and 'H-nmr properties of peptide 401, a bee venom component with 22 amino acid residues and two disulfide bridges, have been studied under a variety of conditions and compared with those of the structurally related octadecapeptide apamin. The major component of the relatively intense CD signal in the 200-230-nm region in both cases probably arises from the rigid asymmetric ring structures of the disulfide bridges. CD spectra are practically unaffected by pH (in the region 1-7), solvent (water, trifluoroethanol, dioxane/water mixtures), concentration of peptide, or additions of salt (guanidinium chloride, KCl). Temperature changes (in the range 20-59°C) have only a modest influence. For both apamin and peptide 401, reduction of the two disulfide bridges results in a dramatic change of the CD spectrum, which acquires the characteristic form of a random coil. Preliminary 'H-nmr data are presented for both the reduced and the oxidized form. Several resonance peaks could be assigned on the basis of the theoretical random-coil spectrum. In the oxidized forms, six slowly exchangeable amide protons could be found in a spectrum taken at low pH, which are ascribed to intramolecular hydrogen bonds. Each of the four protons of the two histidine residues of peptide 401 appears as two distinct resonance peaks in the oxidized form but not in the reduced form. This is interpreted as arising from conformational heterogeneity of peptide 401.
SummaryA conformational study was carried out on Boc-(~-Val-~-Val)~-OMe in the crystalline state by X-ray and IR. and by 'H-NMR. in chloroform. The dodecapeptide crystallizes from CHCl,/EtOAc with a left-handed helical structure of the type TL /?5.6, and from CHCI3/MeOH (or MeOH) with a different structure. In chloroform it forms three slowly interconverting species: one is a ?J p5.6 left-handed helical species, and the other two are most likely single-stranded p4.4 helical species of opposite handedness. The double-stranded helical species is. predominant in fresh solutions of samples obtained from CHCI,/EtOAc. Because of the slow conversion or formation of this species some hours are needed to reach the conformational equilibrium in chloroform at 25 ' .
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