Globoside as a membrane receptor: a consideration of oligosaccharide communication with the hydrophobic domain

Recognition of macromolecules by glycosphingolipids is closely correlated with the nature of the glycolipid carbohydrate; however, it is also thought to be secondarily modulated by the structure of the single fatty acid. In the present work, we sought insight into what physical effect a change in this fatty acid has on the extramembranous portion of globosides at liposomal surfaces mimicking systems for which modulated receptor function has been recorded in the past. Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of globotriaosylceramide (Gb3) were replaced with deuterium. In this location, the nonperturbing probe nuclei sampled cumulative conformational and orientational characteristics of the oligosaccharide chain at a sugar residue that is critical in specific binding of verotoxins. Deuterated Gb3 having 18:1 fatty acid was compared to the same species having 22:1 fatty acid, at 6.3 mol % in unsonicated bilayers of dipalmitoylphosphatidylcholine/cholesterol. Both produced narrow, apparently axially asymmetric 2H NMR spectra over a wide temperature range. Motional properties of the terminal sugar were measurably influenced by the fluidity of the host matrix; however, evidence was not found for conformational or orientational variation in this sugar brought about by the fatty acid alteration. In related experiments, acetate protons on the terminal N-acetyl galactosamine (GalNAc) residue of globotetraosylceramide (Gb4) were substituted with deuterium, and the natural fatty acid was replaced with 18:0 or 24:0 species deuterated at C2. Once again, species with short vs long fatty acid were examined for evidence of headgroup differences. Spectra of Gb4 were compared at 10 mol % in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and at 5 mol % in membranes containing 33 mol% cholesterol. Spectral splittings reflecting cumulative effects on conformation and order at the terminal deuterated sugar remained unchanged between species having 18:0 vs 24:0 fatty acid in POPC/cholesterol. In a pure POPC host matrix, there was clear evidence of a motional difference between the two--the longer chain Gb4 demonstrating spectral asymmetry--but the spectral width was unchanged. Transverse relaxation times, T2, were measured. Our findings appear to help correlate the conclusions of a number of workers dealing with the molecular basis of crypticity. We suggest that changes in glycolipid receptor function based on ceramide fatty acid variation have a major origin in the fatty acid's ability to determine the thermodynamics of interaction with the host matrix, as reflected in such parameters as glycolipid motional properties, local membrane curvature, and likely glycolipid time-dependent lateral associations. The result at low concentrations of glycolipid may often be only a subtly altered collective surface epitope, best detected by a specific recognition event.     

1H-NMR resonance assignments, secondary structure, and global fold of the TR1C fragment of turkey skeletal troponin C in the calcium-free state

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].     

Effect of long chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine on the interaction with recombinant high density lipoprotein apolipoprotein A-I

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.     

Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria

The first three-dimensional structure of a type IIa bacteriocin from lactic acid bacteria is reported. Complete 1H resonance assignments of leucocin A, a 37 amino acid antimicrobial peptide isolated from the lactic acid bacterium Leuconostoc gelidum UAL187, were determined in 90% trifluoroethanol (TFE)-water and in aqueous dodecylphosphocholine (DPC) micelles (1:40 ratio of leucocin A:DPC) using two-dimensional NMR techniques (e.g., DQF-COSY, TOCSY, NOESY). Circular dichroism spectra, NMR chemical shift indices, amide hydrogen exchange rates, and long-range nuclear Overhauser effects indicate that leucocin A adopts a reasonably well defined structure in both TFE and DPC micelle environments but exists as a random coil in water or aqueous DMSO. Distance geometry and simulated annealing calculations were employed to generate structures for leucocin A in both lipophilic media. While some differences were noted between the structures calculated for the two different solvent systems, in both, the region encompassing residues 17-31 assumes an essentially identical amphiphilic alpha-helix conformation. A three-strand antiparallel beta-sheet domain (residues 2-16), anchored by the disulfide bridge, is also observed in both media. In TFE, these two regions have a more defined relationship relative to each other, while, in DPC micelles, the C-terminus is folded back onto the alpha-helix. The implications of these structural features with regard to the antimicrobial mechanism of action and target recognition are discussed.     

2D 1H-NMR conformational study of phosphatidylserine diluted in perdeuterated dodecylphosphocholine micelles. Evidence for a pH-induced conformational transition

The conformation of phosphatidylserine (DMPS) diluted in perdeuterated dodecylphosphocholine micelles (DPC) has been investigated by 1D and 2D proton NMR spectroscopy. Chemical shift pH dependence showed that the pK relative to the serine carboxyl titration (3.4 +/- 0.05) was nearly identical to that measured in bilayers. Chemical shift and NOE data revealed that the phosphatidylserine molecule undergoes a conformational transition upon titration of the serine carboxyl group. The NOE network observed between the different parts of the molecule was sufficiently abundant to allow, in combination with molecular modeling methods, an assessment of the conformational changes. The conformational changes mainly involve the glycerol backbone, which is parallel to the whole molecule, that is, to the layer normal, at low pH and becomes perpendicular to the whole molecule at neutral pH. In both cases, the conformations are remarkably close to those observed for the crystal forms of zwitterionic and negatively charged phospholipids. Two-dimensional proton NMR study of phospholipids, diluted in perdeuterated DPC micelles, appears to be a simple and relevant method to obtain complete and direct information on their conformations in a model membrane-solution interface.     

A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H-, 13C-NMR, and mutational analysis

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.     

Molecular analysis of protein domain function encoded by the myb-homologous maize genes C1, Zm 1 and Zm 38

Two maize genes, Zm 1 and Zm 38, related to the regulatory anthocyanin gene C1 were analyzed molecularly and used for fusion constructs in transient domain swapping experiments with the C1 wild-type gene. It was shown that both genes (Zm 1 and Zm 38) influence the expression of the A1 locus, a target gene for C1. Zm 1 activates the A1 promoter, however it does not turn on the whole anthocyanin pathway. The Zm 38 gene product shows functions similar to C1-I, a dominant inhibitor of the C1 wild-type gene. Concerning the trans-inhibition by C1-I two effects seem to be involved, competition for binding and formation of heterodimers. Further analysis of C1 function was carried out by a fine structure analysis of C1 mutants induced by the insertion and excision of transposable elements. These experiments indicate that for the activating domain of the protein, the formation of an alpha helix seems to be more important than a high negative charge.     

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.     

The diagnostic capacity of serum amyloid A protein for early recognition of kidney allograft rejection

Chitosan derivatives, sulfated N-acyl-chitosan (S-Cn-chitosan) possessing various lengths of alkyl chain, were prepared, and the properties of their aqueous solutions were examined. The 1H-NMR spectrum of D2O solutions of S-C12-chitosan showed broadening of the proton signals caused by aggregation of the alkyl chain. The solubility of a hydrophobic compounds, azobenzene, was small in the aqueous solutions of S-Cn-chitosan with shorter alkyl chains, but increased with increasing length of the chains above C10, showing that micelles had been formed. The ESR spectrum of a spin probe, TEMPO, in an S-C14-chitosan solution showed the existence of a hydrophobic region in the solution, but this region did not exist in the S-C2-chitosan solution. The rigidity of this region was examined by using a spin probe, 16-doxyl-stearic acid. From these results, it was revealed that S-Cn-chitosan with longer alkyl chains formed a novel type of micelle called a "polymer micelle," which was more stable than the ordinary micelles formed from low-molecular-weight surfactants.     

Deuteriation of sugar protons simplify NMR assignments and structure determination of large oligonucleotide by the 1H-NMR window approach

The concept of the 1H-NMR window has been developed and examined through a comparative study of NOESY spectra of a self-complementary Dickerson's dodecamer (I) [5'd(5C6G7C8G9A10A11T12T13C-14G15C16G)2(3')], a self-complementary 20-mer (II) [(5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core part consists of the same Dickerson's dodecamer sequence with the flanking CGCG residues at both 3' and 5'-ends, and the partly-deuteriated (shown by underlined CGCG residues at both 3' and 5'-ends) analogous duplex (III) [5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core 5C to 16G part (i.e. 1H-NMR window) consists of the natural Dickerson's dodecamer sequence. A comparison of their NOESY spectra clearly demonstrates that the severe overlap of proton resonances in the larger DNA duplex (II) has been successfully reduced in the partly-deuterated duplex (III) as a result of specific incorporations of the sugar-deuteriated nucleotide residues in the latter [stereospecific > 97 atom % 2H enrichment at H2', H2' and H3' sites, approximately 85 atom % 2H enrichment at H4' and approximately 20 atom % 2H enrichment at H1' (see refs. 10 and 11) in the 20-mer duplex (III)]. These simplifications of the resonance overlap by the deuteriation approach have enabled unequivocal chemical shift assignments and extraction of the quantitative NOE data in the 1H-NMR window part of duplex (III). A comparison of the 12-nucleotide long 1H-NMR window in (III) with that of the 12-mer duplex (I) also shows the scope of studying the changes in conformation and dynamics of the core 12-mer region in (III) which result from the increase of molecular weight due to the DNA chain extension. It is noteworthy that such a study is clearly impossible for the natural 20-mer (II) because of the inherent problem of the overlap of resonances.     

Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide

The molecular organization and dynamics have been investigated in membranes consisting of 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) and various ratios of a transmembrane alpha-helical peptide, Ac-K2L24K2-amide (L24), in order to gain insights into how the transmembrane portions of membrane proteins are mixed with phospholipids and organized in biological membranes. Particular attention was paid to membranes with high peptide concentrations. The molecular organization and dynamics were studied in the ps-to-micros regime using various spin-labeling techniques. Conventional ESR spectra as well as saturation-recovery curves measured in both the presence and the absence of molecular oxygen showed that phosphatidylcholine spin-labels detect the existence of a single homogeneous environment, indicating that both L24 and POPC are likely to be undergoing fast translational diffusion in L24-POPC membranes of up to 9 mol % peptide. Since 16-18 molecules of phosphatidylcholine are required to surround a transmembrane alpha-helical peptide [Morrow, M. R., Huschilt, J. C., and Davis, J. H. (1985) Biochemistry 24, 5396-5406], L24 must form L24-rich regions at a P/L ratio of 1/10 instantaneously. However, these results suggest that the lipid exchange rates among the bulk, boundary, and L24-rich regions are fast, and that the L24-rich regions must be forming and dispersing rapidly in a time scale shorter than 0.1 micros, the conventional ESR spin-label time scale and the electron spin-lattice relaxation time scale in the presence of molecular oxygen. Although this does not exclude the possibility of the formation of small, stable oligomers of L24, it is unlikely because L24 lacks features that would favor their formation. L24 (9 mol %) increases the hydrophobicity of the central part of the POPC membrane from the level of 1-decanol to that of pure hexane and also increases the hydrophobicity near the membrane surface from the level of 2-propanol to that of 1-decanol. The effect of 9 mol % L24 on the order parameter profile is similar to that of decreasing the temperature by approximately 8 degrees C between 10 and 55 degrees C. It is concluded that L24 is highly miscible in POPC membranes even at high concentrations in the membrane.     

PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: transcriptional regulation by TGF-beta 1

The thermally sensitive block copolymer, poly(N-isopropylacrylamide-b-dl-lactide) (PIPAAm-PLA), was synthesized by ring-opening polymerization of dl-lactide initiated from hydroxy-terminated poly (N-isopropylacrylamide) (PIPAAm). A PIPAAm bearing a single terminal hydroxyl group was prepared by telomerization using 2-hydroxyethanethiol as a chain-transfer agent. Successful preparation of PIPAAm and the PIPAAm-PLA block copolymer was verified by gel permeation chromatography (GPC) and 1H-NMR spectroscopy. Polymeric micelles were prepared from block copolymers using a dialysis method. Their solutions showed reversible changes in optical properties: transparent below a lower critical solution temperature (LCST) and opaque above the LCST. Dynamic light scattering measurements were used to observe the formation of micellar structures approximately 40 nm in diameter, which do not change between 20 degreesC and 30 degreesC. Above the LCST, polymer micelles aggregated, a phenomenon found to be reversible since the aggregates dissociated again by cooling below the LCST. Further observations using atomic force microscopy (AFM) confirmed this behaviour. The properties of this block copolymer system are interesting from both applied and fundamental perspectives, particularly for active targeting as drug carriers.     

Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.     

Effect of cholesteryl hemisuccinate on the interfacial properties of phosphatidylcholine bilayers

Cholesteryl hemisuccinate (CHEMS) is an amphipathic lipid that can regulate cell growth. A comparison of the effects of CHEMS and cholesterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers was investigated using fluorescence techniques. In liquid-crystalline phase POPC bilayers, CHEMS increased the interfacial surface charge, but was less effective than cholesterol in reducing acyl chain mobility and interfacial hydration. In liquid-crystalline phase DPPC bilayers, CHEMS and cholesterol were equally effective in reducing acyl chain mobility. Similar to the POPC matrix, CHEMS increased the interfacial surface charge and cholesterol decreased the surface hydration. The different effect of cholesterol and CHEMS on acyl chain mobility may be due to a preferential interaction of cholesterol with POPC. In gel phase DPPC bilayers, CHEMS and a succinylated pyrenyl cholesterol analog exhibited different effects on membrane physical-chemical properties than cholesterol. Succinylation also increased the rate of transfer of the pyrenyl cholesterol analog between single unilamellar vesicles approximately seven fold. This process demonstrated first-order kinetics which indicated that transbilayer migration was not a rate-limiting step. The succinylation of cholesterol places a carboxyl group at the lipid-water interface and the sterol ring deeper in the bilayer. For a structural model to explain its biological properties, CHEMS should be considered a bulky fatty acid.     

Video-assisted theloscopic electroincision of a high teat stenosis

The hereditary neuropathy Charcot-Marie-Tooth (CMT) type 1A is, in the majority of cases, caused by duplication of the gene for the peripheral myelin protein PMP22, which leads to abnormally increased PMP22 expression. Recent in vitro and in vivo data indicate a novel function of PMP22 in Schwann-cell growth and differentiation other than its role in myelination, and suggest that overproduction of PMP22 leads to a new Schwann-cell phenotype in CMT1A. Taking these data into account, we developed a new hypothesis on the pathogenesis of CMT1A neuropathy: that the defective myelin stability and turnover observed in the disease is caused by altered PMP22 gene dosage and its resultant effect on abnormal Schwann-cell growth and differentiation.     

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Basic structure

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic-interaction chromatography. The preparations from both strains have an identical structure: a 1,3-linked poly(glycerophosphate) chain phosphodiester-linked to Glc-(alpha 1-2)Glc(alpha 1-3)acyl2Gro as the lipid anchor. The chain is substituted with D-alanine ester and glycosyl residues which comprise mono-, di-, tri- and tetra-alpha-D-glucopyranosyl residues with (1-6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H-NMR and 13C-NMR spectroscopy. The alpha-isomalto-oligosaccharides add a novel motif to lipoteichoic-acid chain substituents. 1H-NMR and 13C-NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3-glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor. Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).     

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.     

1H-NMR studies of the ACTH-like immunoregulatory peptides

A comparative study of the conformational and dynamics properties of the ACTH-like linear peptides, sequences of which correspond to amino acid residues 11-20 of the heavy chain of human immunoglobulin G1 Eu, residues 78-85 of human pro-interleukin-1 alpha and site 10-18 of human ACTH, was performed in aqueous solution and dimethylsulfoxide by 1H-NMR spectroscopy at 400 MHz. The peptides were shown to possess an unordered unfolded flexible conformation in aqueous solution. The revealed structural and dynamic features of the peptides are discussed together with biological activity of this class of compounds.