1H-n.m.r. studies on cross-relaxation phenomena in bovine mercaptalbumin (BMA) solution and partially hydrolyzed bovine plasma albumin (BPA*) gel

Bovine plasma albumin Fr. V(BPA) contains small amounts of proteolytic enzyme which catalyzes a very limited cleavage of BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA(BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 7%. Highly purified proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12.4%. after 5 days incubation at 35°. Cross-relaxation times (T_IS) between irradiated protein protons and observed protons, such as side chain and water protons, were studied on BMA solution and BPA*-gel. T_IS values of BMA solution, obtained by the saturation transfer (SATUR) and inversion recovery (INVER) methods, were a single kind of T_IS for each side chain. Those of BPA*-gel by the SATUR method indicated the presence of two kinds of T_IS, that is, short and long T_IS values for each side chain. However, those by the INVER method showed a single kind of T_IS for each side chain, corresponding to the long T_IS value by the SATUR method. The short T_IS values of BPA*-gel, observed by the SATUR method, may be due to immobile joint parts of fibrous BPA* aggregates. T_IS values from protein to water protons (T_IS(HDO)) in BPA*-gel, obtained by the INVER method, were far shorter than those in BMA solution, indicating a large amount of hydration of BPA* and rapid exchange between bound and bulk water in the gel state.     

Conformational studies on gastrin related peptides by high resolution 1H-n.m.r.

The secondary structures of three gastrin analogs, HCl · H-Trp-Nle-Asp(O-tBu)-Phe-NH₂ (tetragastrin), pGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH₂ (octagastrin), and H-Leu-(Glu)₅-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH₂ (minigastrin) were studied by ¹H-n.m.r. in dimethylsulfoxide and in trifluoroethanol. All three compounds were found to assume a random conformation in the former solvent, while some ordered secondary structure is present in trifluoroethanol even at the tetra-peptide level. This was shown by temperature studies and solvent titrations. At least four amide protons were found to be solvent shielded in the longer hormone.     

1 H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A

The reaction of ribonuclease A with either 6-chloropurine riboside 5′-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the α-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of ¹H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3′AMP is similar to that described for RNase A as the pK₂ of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK₂ values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK₂ of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.     

1H-n.m.r. studies on the specificity of the interaction between bovine pancreatic ribonuclease A and dideoxynucleoside monophosphates

The titration curves of the C-2 histidine protons of bovine pancreatic ribonuclease A in the presence of several dideoxynucleoside monophosphates (dNpdN) were studied by means of proton nuclear magnetic resonance at 270 MHz in order to obtain information on the ligand — RNase A interaction. The changes in the chemical shift and pKs of the C-2 proton resonances of His-12, -48, -119 in the complexes RNase A — dNpdN were smaller than those previously found when the enzyme interacted with mononucleotides. The pK₂ of His-12 was not affected by the interaction of the enzyme with these ligands, whereas, the perturbation of the pK₂ of His-119 was clearly dependent on the nature of the ligand. If there is a pyrimidine nucleoside at the 3′ side of the dideoxynucleoside monophosphates, as in TpdA and TpT, an enhancement due to the well known interaction of the phosphate in p₁, the catalytic site, was found. However, when there is a purine nucleoside, as in dApT and dApdA, a decrease in the pK₂ value was observed and we propose that in such cases the phosphate group interacts in a secondary phosphate binding site, p₂. The results obtained suggest the existence of different specific interactions depending on the structure of the dideoxynucleoside monophosphate studied.     

Controlled proteolysis of mouse epidermal growth factor An RP-HPLC and 1H-n.m.r. study

Tryptic digestion of the mouse epidermal growth factor (mEGF) and the chromatographic separation of its proteolytic fragments by RP-HPLC affords the isolation of the pure hormone, of its 1–48 (Des(49–53)mEGF) and 1–45 (Des(46–53)mEGF) derivatives, and of the carboxyl-terminal pentapeptide W49–W50-E51-L52-R53. Kinetics of mEGF proteolytic degradation follows a two-state time-course: native mEGF being converted into Des(49–53)mEGF with an apparent half-time of 10min; and Des(49–53)mEGF subsequently hydrolyzed to Des(46–53)mEGF with an apparent half-time of 7 h. Native mEGF and its proteolytic fragments have been characterized by ¹H-n.m.r. spectroscopy. In the aromatic and aliphatic regions, the ¹H-n.m.r. spectrum proved to be a sufficiently sensitive probe for following controlled proteolysis, and for analyzing the influence of the carboxyl-terminal sequence on the hormone conformation and stability.     

CD and 1 H-n.m.r. studies on the side-chain conformation of tyrosine derivatives and tyrosine residues in di- and tripeptides

Tyrosine, tyrosine peptides and derivatives, in total 11 species, were selected as models for the study of optical properties (¹L_b band of phenolic group) and side-chain arrangement (rotamers around C_α – C_β bond) of tyrosine as a function of chemical structure and pH effects. Circular dichroism spectra between 240 and 320 nm and n.m.r. spectra were recorded for the different ionization states. Results are discussed in terms of charge effects from N- and C-terminal groups and local conformation influence on ¹L_b band of the phenolic chromophore and on distribution of rotamer populations in side-chain of tyrosine. Fractions of rotamer populations were estimated from α-β proton-proton coupling constants and, in the cases of tyrosine and N-acetyl-tyrosine, from ¹⁵N-β nitrogen-proton coupling constants, which allow the stereospecific assignment of the β and β′ protons. The rotamer populations of tyrosine, averaged from all the data of the samples in solution, were then compared with their statistical distribution in the solid state. Interestingly, agreement is excellent when we refer to crystal of tyrosine, tyrosine derivatives or small peptides (31 samples) and poor in the case of proteins. This leads to a discussion on both the validity of using statistical distributions of rotamers in proteins as reference for rotamer preferences inside small peptides in solution and the choice of the appropriate J_g and J_t values in Pachler's approach. The possible existence of a correlation between ellipticity and rotamer populations for such samples is examined.     

Sodium dodecyl sulfate-bovine plasma albumin complex

The acid-induced isomerization (the N-F transition) and expansion of sodium dodecyl sulfate-bovine plasma albumin complex (AD_m;m, molar ratio of added sodium dodecyl sulfate to bovine plasma albumin; 0 ± m ± 12) were studied by measuring CD-resolved secondary structure, fluorescence polarization and lifetime of tryptophyl fluorophors, acid-titration with the electrostatic correction for the surface potential, ¹H-n.m.r. spectra and cross relaxation time between irradiated and observed protons. The immobilization of tryptophyl fluorophors observed in the F-form of AD₀ was suppressed in the F-form of AD₁₀. The acid-titration analysis of AD₁₂ showed non salt-bonding between carboxylate groups and cationic side chains in the F-form, as in the case of AD₀, indicating charged side chains being presumably mobile. ¹H-n.m.r. spectra and cross relaxation times between irradiated and observed protons in the F-form of AD₁₀ indicated the increase in the local motion. On the other hand, AD₁₀ and AD₁₂ did not show any significant change in the CD-resolved secondary structure in the N-F transition region. The F-form of AD₁₀ or AD₁₂ may therefore be the molten-globule state which has secondary structure similar to the N-form of the complexes with fluctuating tertiary structure (side chains).     

SMS 201–995, a very potent analogue of somatostatin. Assignment of the 1H 500 MHz n.m.r. spectra and conformational analysis in aqueous solution

The conformations of a cyclic analogue of somatostatin, SMS 201–995, have been studied by n.m.r. spectroscopy at 500 MHz in aqueous solution. Assignments were made by use of 2D-correlated methods, especially by detecting long-range connectivities in order to identify the aromic amino-acid and long-range couplings between α protons of consecutive residues. Measurements of temperature coefficients of amide protons and of NH-C_αH coupling constants enabled us to conclude that in water the molecule is rather flexible, with no evidence for a β turn structure involving Thr⁶. An equilibrium involving two γ turn conformations stabilized respectively by Cys²-d -Trp⁴and Phe³-Lys⁵hydrogen bonds, is responsible for the large upfield shift observed for the Lys⁵γ protons and is compatible with the measured J_NH-Cα_H coupling constants.     

1H-n.m.r. study of the folding of ribonuclease 12-(β-(3-pyridyl)-l-Ala) S-peptide (1–14)

The ¹H-n.m.r. spectra (360 MHz) of 12-(β-(3-pyridyl)-l -Ala) ribonuclease S-peptide (1–14), a tetradecapeptide incorporating (β-3-pyridyl-l -Ala) instead of His at position 12, have been assigned. The shift vs. temperature dependence has been analyzed at three different pD's in terms of a two-state helix (3–13) ± coil equilibrium, and the corresponding values for the thermodynamic quantities ΔH° and ΔS° determined. Helix populations at 0°C have been measured as a function of pD, showing their dependence on two apparent pK_a's at ? 3.3 and 5.5, with a maximum at pD ? 4.2. All the obtained results show that the new peptide has very similar folding properties to those shown by S-peptide and particularly to those of C-peptide. The 3–13 helix formed is stabilized by two interactions: a salt-bridge Glu 2^-. Arg 10+ and a partial stacking between the aromatic rings of residues Phe 8 and His 12. Calculations involving ring current shifts and potential energies validate the possible existence of this latter interaction, which must present a local geometry defined by χ₁X⁸ 180°, χ₂X⁸ 100°, χ₁¹² – 60 and χ₂¹² 80.     

Isolation,purification and 13C-and 1H-n.m.r. assignments of peptide [1–24] of human serum albumin

Isolation, purification and 360 MHz ¹H- and ¹³C-n.m.r. spectra of the residue corresponding to the NH₂-terminal peptide fragment [1–24] of human serum albumin are reported. The various resonances have been assigned to individual amino acid residues and their spatial microenvironment has been determined in a straightforward manner on the basis of (i) pH dependent chemical shifts; (ii) combined use of multiple and selective proton-decoupled ¹H- and ¹³C-n.m.r. spectra; (iii) the characteristic pK values exhibited by protons adjacent to sites of ionization in the molecule; and (iv) comparison of the spectra with the NH₂-terminal tripeptide segment of human albumin. The pK values of different ionizable groups all fall in the normal range expected for each titrating sites and support a model of peptide fragment [1–24] in which there is no special structure-forming strong associations. These results are in agreement with those obtained by CD spectroscopy.     

1H-NMR and CD studies on the structural transition of serum albumin in the acidic region—The N→F transition

Helical content (f_α) of bovine mercaptalbumin (BMA) showed the characteristic two-step decrease in the acidic region, one corresponding to the N→F transition (pH 4.40→3.75; f_α, 0.68→0.58) and the other to the F→E transition (the acid-expansion) (pH 3.60→2.90; f_α, 0.58→0.48). However, f_α of human serum albumin (HSA) mainly decreased in the N→F transition (N→F, pH 4.6→3.4; f_α, 0.70→0.55 and F→E, below pH 3.0; f_α, 0.55→0.52). The difference in pH-profile of f_α between BMA and HSA might be due to the microheterogeneity. The ¹H-NMR spectra and cross-relaxation times (T_IS) from irradiated to observed protein protons, which reflect the structural fluctuation and/or mobilability in proteins, were measured on the N-, F-, E-forms of HSA and BMA, and the N*-form (8.23 M urea, neutral pD) of iodoacetamide-blocked HSA (IA-HSA) and bovine serum albumin (IA-BSA). The ¹H-NMR spectra and elongations of T_IS values for the F- and E-forms of HSA and the E-form of BMA were quite similar to those for the N*-form of IA-HSA and IA-BSA, indicating the liberation of the intramolecular motion in the F- and E-forms. Those for the F-form of BMA were intermediate between the N- and E-form. The present results together with the reported data on hydrodynamic radii and D–H exchange reaction, indicate that the F-form of HSA and presumably BMA has a native-like globule form with a highly helical state and fluctuating tertiary structure. Thus, all of the present findings on the F-form of serum albumin seem to be in accord with the structural features for the F-form suggested by Foster's group (1-3, 19, 20, 22, 23) and the molten globule state demonstrated by Dolgikh et al. (40), and Ohgushi and Wada (36, 37).     

Comparison of conformational properties of linear and cyclic δ selective opioid ligands DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) and DPLPE (Tyr-c[D-Pen-Gly-Phe-Pen]) by 1H n.m.r. spectroscopy

The preferential conformations of the δ selective opioid peptides DPLPE (Tyr-c[D-Pen-Gly-Phe-Pen]) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) were studied by 400 MHz ¹H n.m.r. spectroscopy in DMSO-d₆ solution. In neutral conditions, the weak NH temperature coefficients of the C-terminal residue (Pen⁵ or Thr⁶), associated with interproton NH-NH and α-NH NOE's (ROESY experiments), indicated large analogies between the backbone folding tendency of both the linear and cyclic peptides. Various γ and/or β turns may account for these experimental data. A similar orientation of the N-terminal tyrosine related to the folded backbones is observed for the two agonists, with a probable γ turn around the amino acid in position 2. Finally, a short distance, about 10 Å, between Tyr and Phe side chains and identical structural roles for threonyl and penicillamino residues are proposed for both peptides. These results suggest the occurrence of similar conformers in solution for the constrained peptide DPLPE and the flexible hexapeptide DTLET. Therefore, it may be hypothesized that the enhanced δ selectivity of DPLPE is related to a very large conformational expense of energy needed to interact with the μ opioid receptor, a feature not encountered in the case of DTLET. These findings might allow peptides to be designed retaining a high affinity for δ opioid receptors associated with a very low cross-reactivity with μ binding sites.     

17O, 14N and 15N n.m.r. studies of the CO2+ complexes of cyclo(Pro17O-Gly15N) and cyclo(Gly17O-Pro) in aqueous solution

The complexation of cyclo(Pro¹⁷O-Gly¹⁵N) and cyclo(Gly¹⁷O-Pro) with CO²⁺ ions has been studied by ¹⁷O, ¹⁴N and ¹⁵N n.m.r. spectroscopy in aqueous solution. ¹⁷O, ¹⁴ N and ¹⁵N transverse relaxation times and chemical shifts were measured as a function of temperature. The ¹⁷O n.m.r. studies unequivocally demonstrate that the cobaltous ion binds to the peptide oxygen of both compounds. The hyperfine coupling constant and the peptide residence times were found to be A =–0.165 MHz and – 0.145 MHz, μ= 16, and 92μsec for cyclo(Pro¹⁷O-Gly¹⁵N) and cyclo(Gly¹⁷O-Pro), respectively. The ¹⁴N and ¹⁵N studies of labeled cyclo(Pro¹⁷O-Gly¹⁵N) do not indicate binding at either the Gly¹⁵N or the Pro¹⁴N site.     

1 H n.m.r. long-range coupling in 2,4-disubstituted-5(4H)-oxazolones and 4-alkyl-5(2H)-oxazolones generated therefrom by the action of triethylamine

Long-range couplings were observed between H-4 and 2-CCH_a of 2,4-disubstituted-5(4H)-oxazolones, and H-4 and H-2 of 4-alkyl-5(4H)-oxazolones. In the presence of triethylamine, H-4 of the latter migrates to C-2 accompanied by a shift of the double bond to give 4-alkyl-5(2H)-oxazolones which show ⁵J coupling between H₂-2 and 4-CCH_a protons.