Analysis of the 1H-NMR spectra of ferrichrome peptides. II. The amide resonances

The high-resolution ¹H-nmr study of the ferrichrome cyclohexapeptides, in d6 -DMSO solutions, has been extended to the amide NH spectral region. A total of ten diamagnetic analogues of ferrichrome that differ in the coordinated metal ion (Al³⁺, Ga³⁺ or Co³⁺), the primary structure, the nature of the bidentate hydroxamate moiety, or the isotope compositions (¹⁴N, ¹⁵N) have been investigated. The ³J_αNH values reflect regiorous conformational isomorphism throughout the complete suite of analogues, quite independent of the residue occupancy of each site. Totally resolved amide multiplets have been obtained in most cases and the four-line (doublet of doublets) appearances of glycyl NH resonances has been observed for the first times; these data enabled stereospecific assignment and accurate analysis of the NH-CαH proton spin systems. The high resolution was made possible by the use of a suitable spectral deconvolution shceme at 360 MHz. The fine structure, extraordinarily well displayed in the ¹⁵N-peptide spectra, provides a series of parameter values whose consistency has been checked by computer simulation. Since the crystallographic structure for two of the ferric peptides is known to 0.002-Å resolution, a ³J vs θ correspondence could be confidently established. A Karplus curve was derived from the combined x-ray and nmr data: It is suggested that seriously nonplanar amides can exhibit ³J_αNH values higher than predicted by the ferrichrome curve.     

Structural analysis of peptides capable of binding to more than one Ia antigen

The Ia binding regions were analyzed for three unrelated peptide Ag (sperm whale myoglobin 106-118, influenza hemagglutinin 130-142, and lambda repressor protein 12-26) for which binding to more than one Ia molecule has previously been demonstrated. By determining the binding profile of three separate series of truncated synthetic peptides, it was found that in all three cases the different Ia reactivities mapped to largely overlapping regions of the peptides; although, for two of the peptides, the regions involved in binding the different Ia specificities were distinct. Moreover, subtle differences were found to dramatically influence some, but not other, Ia reactivities. Using a large panel of synthetic peptides it was found that a significant correlation exists between the capacity of peptides to interact with different alleles of the same molecule (i.e., IAd and IAk), but no correlation was found with the capacity of peptides to interact with different isotypes within the same haplotype (i.e., IAd and IEd). These data suggest that different alleles of the same MHC molecule may actually recognize closely related structures, whereas different isotypes may recognize unrelated, albeit non-mutually exclusive, structures on an Ag molecule.     

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Evidence for more than one division of bacteria within airborne particles.

When the protocol that we had used to demonstrate a single division of bacterial cells in airborne particles was changed to one that increased the glycerol content of the atomizer fluid from 1 to 5% (vol/vol), thus producing larger particles, more than two (and nearly three) divisions of bacteria occurred within 6 h of aerosol time.     

Unidirectional fluxes in saturated single-file pores of biological and artificial membranes I. Pores containing no more than one vacancy

A theoretical treatment of isotope fluxes in filled single-file pores is given. The treatment is confined to pores permitting only one vacancy at a time. Tracer flux, unidirectional fluxes and net flux are calculated. The exponent n which is obtained by representing the ratio of unidirectional fluxes as a power of the electrochemical activity-ratio proves to be closely related with m, the number of sites per pore. The minimum and maximum values of n are m?1 and m. It is shown that measurement of n provides sufficient information to determine m if the energetic properties of sites and barriers do not change too much with varying concentrations. Unlike the unidirectional fluxes, the net fluxes yield no direct information about the number of sites. The concentration dependence of the net fluxes, however, can be used to discriminate between the two limiting cases, n = m?1 and n = m. It is interesting with regard to the instantaneous potassium currents of nerve membranes that a suitable choice of the energetic parameters of a filled pore leads to a quasi-linearity of the net flux-voltage curves, which is only slightly affected by variation of the external concentration.     

Evidence for more than one division of bacteria within airborne particles.

When the protocol that we had used to demonstrate a single division of bacterial cells in airborne particles was changed to one that increased the glycerol content of the atomizer fluid from 1 to 5% (vol/vol), thus producing larger particles, more than two (and nearly three) divisions of bacteria occurred within 6 h of aerosol time.     

Binding of more than one retinoid to visual opsins

Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.     

Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes.

The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.