IR (vibrational) CD of peptide beta-turns: a theoretical and experimental study of cyclo-(-Gly-Pro-Gly-D-Ala-Pro-).

IR vibrational CD (VCD) has been observed for the cyclic pentapeptide cyclo-(-Gly-Pro-Gly-D-Ala-Pro-) in solution in CDBr3. The observed VCD spectra do not resemble the VCD features of any of the previously reported peptide secondary structures, such as alpha-helical, "random coil," or sheet structures, and might be due to the beta-turn contained in this molecule. To shed light onto the origin of the observed spectra, VCD intensity calculations, based on the solution and solid-state structures of cyclo-(-Gly-Pro-Gly-D-Ala-Pro-), have been carried out. In addition, calculated VCD data for pure beta-turns are discussed.

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.

The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms.

Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.

Synthesis and biological activities of a photoaffinity probe for vasotocin receptors.

This study reports the synthesis and biological activities of 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT). This compound was found to be biologically active in the rat antidiuretic assay (20 U/mg), to behave as an antagonist of vasopressin in the rat pressor assay (pA2 = 6.6), and to yield a half-maximal hydroosmotic response in the isolated toad urinary bladder at a bath concentration of 2.4 X 10(-8) M. When toad bladders were exposed to d7-N3-AVT in the presence of long wavelength UV light, the hydroosmotic response persisted in spite of prolonged and repeated periods of washout. By contrast, the hydroosmotic response in control bladders after stimulation with d7-N3-AVT in the absence of UV irradiation was fully reversed within 15 min of washout. A membrane preparation derived from bladders that had been photolabeled with d7-N3-AVT and washed for 1 h specifically bound 325 fmol [3H]vasopressin/mg protein. Matched bladders exposed to the analog in the absence of UV irradiation and washed for 1 h specifically bound 591 fmol [3H]vasopressin per mg of protein. These studies indicate that d7-N3-AVT binds covalently to hydroosmotic receptors of toad urinary bladder and forms a complex that is functional in triggering an increase in the permeability to water of the epithelium. This analog may prove useful in the isolation and purification of vasotocin receptors in lower vertebrates.

Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a protein inhibitor of calcium phosphate precipitation in human saliva.

Human salivary secretions are supersaturated with respect to basic calcium phosphates but spontaneous precipitation of these salts from saliva, or surface-induced precipitation of calcium phosphates onto dental enamel, does not normally occur. This unexpected stability has been attributed to the inhibitory activities of two kinds of salivary phosphoproteins: statherin and the acidic, proline-rich phosphoproteins (PRP). Investigation of the structure-function relationships of statherin, the most potent inhibitor of primary (spontaneous) and secondary (seeded) precipitation of calcium phosphate salts in human saliva has been limited to studies of peptide segments obtained from the native peptide by specific proteolysis. Solid phase peptide synthesis (SPPS) is a useful and potentially more flexible alternative. Phosphoserine residues (positions 2 & 3) play critically important roles in the precipitation-inhibition activities of statherin, but SPP synthesis of these phosphorylated peptides is precluded because of the instability of phosphoserine residues in the presence of HF. Thus, this peptide was synthesized by solution-phase methods. The dipeptide possessed substantial inhibitory activity in assays for inhibition of both primary and secondary precipitation of calcium phosphate salts, but was not as active as either N-terminal tryptic hexapeptide of statherin or intact statherin. Syntheses of other model phosphorylated peptides are underway to expand the structure-function relationships.

Synthesis and biological activities of arginine-vasopressin analogues with 4-hydroxyproline in position 7.

Three arginine-vasopressin (AVP) analogues in which the proline residue in position 7 was substituted with 4-hydroxyproline were synthesized by solid-phase techniques, and their biological activities were evaluated by antidiuretic, pressor, and uterotonic bioassays. The [7-trans-4-hydroxy-L-proline]AVP, the 1-desamino[7-trans-4-hydroxy-L-proline]AVP, and the 1-desamino[7-cis-4-hydroxy-L-proline]AVP analogues showed a high antidiuretic and strikingly high uterine activity, a sharp decrease in pressor activity, and a better antidiuretic and uterine to pressor selectivity than the parent compound, arginine-vasopressin. The uterine activities are the highest so far assayed in AVP analogues with replacements in position 7.

[1-Desamino, 7-lysine, 8-arginine]vasotocin: attachment of reporter groups and affinity ligands through the lysine side chain.

The chemically reactive groups of [8-arginine]vasotocin (AVT) are the alpha-amino group in position 1, the phenolic hydroxyl group of the tyrosyl residue in position 2, and the side-chain functional group of the basic amino acid residue in position 8. Acylation or alkylation of any of these chemically reactive groups yields hormone analogues with sharply diminished biological activities in most assay systems. Since none of the chemically reactive groups in the native AVT (or other neurohypophyseal hormone) sequences is a suitable chemical port for acylation with affinity ligands and reporter groups, we have undertaken the rational design of AVT analogues in which a residue capable of being acylated has been incorporated into points of the AVT structure where structural modifications are expected to have as little effect as possible on biological activity. Empirical structure-activity relationships among neurohypophyseal hormone analogues as well as conformational models in solution and in the crystalline state suggest that positions 4 and 7 are likely points for the introduction of acylation ports. We have previously synthesized an analogue of [1-desamino]AVT (dAVT) with a lysyl residue in position 4 ([4-lysine]dAVT) and demonstrated that acylation of the side chain of this residue yields useful reporter and photoaffinity analogues. We now report the synthesis of the corresponding analogue with a lysyl residue in position 7 ([7-lysine]dAVT), which also yields potent acyl derivatives suitable for affinity ligands and photoaffinity ligands.

Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin.

A photoreactive analogue of vasotocin, [1-desamino,4-lysine(azidobenzoyl),8-arginine]vasotocin (4-N3-AVT), has been examined in the isolated toad urinary bladder for biological activity and binding to hormonal receptors. Although 4-N3-AVT induced only a small increase in bladder permeability to water, it behaved as a potent inhibitor of hydrosmotic action of [8-arginine]vasotocin (AVT) and [8-arginine]vasopressin (AVP). The inhibitory action of 4-N3-AVT was readily reversed on removal of the analogue from the serosal bathing solution. On the other hand, when bladders were exposed to 4-N3-AVT in the presence of long wavelength UV light (365 nm), the inhibition by 4-N3-AVT was not reversed on washout of the analogue. The dose of vasopressin required for a half-maximal response (ED50 value) was increased from 5 X 10(-9) to 1.3 X 10(-7) M in bladders photolabeled with 4-N3-AVT and the maximal response capacity of the tissue (intrinsic activity) was reduced to 79% of nonphotolabeled controls. A crude membrane preparation derived from bladders photolabeled with 4-N3-AVT contained 72 fmol of specific binding sites for tritium-labeled vasopressin per milligram protein, whereas nonphotolabeled controls had 136 fmol of specific binding sites per milligram protein. These observations suggest that 4-N3-AVT forms a covalent bond with hydrosmotic receptors in the presence of UV light. This is the first antagonistic photoaffinity analogue observed in the toad bladder and it may serve as a useful tool for analyzing the cellular mechanism of action of antidiuretic hormone.

Permeability characteristics of the guinea pig biliary apparatus.

To determine the mechanism and pathway of entry of polar nonelectrolytes into bile, we studied first the permeation of [3H]H2O, [14C]urea, [14C]erythritol, [14C]mannitol, [3H]sucrose, [3H]inulin and [3H]dextran across an isolated, in situ perfused segment of the guinea pig's extrahepatic bile duct. All of these molecules, except [3H]dextran, permeated the bile duct. The diffusive permeability coefficients (cm per sec per 10(6) ranged from 248 for [3H]H2O to 1.2 for [3H]inulin. On the basis of these results, we formulated two models of solute biliary excretion. In Model I, permeation across both the canaliculus and ductule/duct was assumed to be by simple diffusion. In Model II, solute was assumed to enter the canaliculus by convection and diffusion, and the ductule/duct by diffusion. Reflection coefficients and/or permeability coefficients for the canalicular membrane were then determined by fitting the equations describing these processes to the bile-to-plasma ratios of [14C] erythritol, [14C]mannitol, [3H]sucrose and [3H]inulin observed at different rates of bile flow produced by partially clamping the bile duct cannula and subsequently infusing taurodehydrocholate. Only when convection was included as a mechanism of canalicular permeation (Model II) was excellent fitting observed. In such a case, the reflection coefficients and permeability coefficients for the carbohydrates in question were similar to those reported for other transporting epithelia. Cholestasis produced by taurolithocholate (10 to 40 mumoles per kg, i.v.) was associated with an irreversible increase in both the sieving coefficient and permeability coefficient for [3H]sucrose and [3H]inulin, even when the inhibition of bile flow was fully reversible. The permeability to [14C] erythritol and [14C]mannitol was either not affected or minimally increased. These findings suggest that, in the guinea pig: solutes as large as [3H]inulin enter the biliary tree both at the canaliculus and bile ductule/duct; [14C]erythritol and [14C]mannitol gain access into the canalicular lumen primarily by convection, whereas [3H]sucrose and [3H]inulin permeate mainly by diffusion; distal permeation of these carbohydrates is small, and accounts for 1 to 11% of their total biliary entry; the canalicular membrane permeability to [3H]sucrose and [3H]inulin is not functionally important in bile secretion, and a fraction of canalicular bile flows through the transjunctional shunt pathway.

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics.

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.

Time-resolved fluorescence and 1H NMR studies of tyrosyl residues in oxytocin and small peptides: correlation of NMR-determined conformations of tyrosyl residues and fluorescence decay kinetics.

Steady-state and time-resolved fluorescence properties of the single tyrosyl residue in oxytocin and two oxytocin derivatives at pH 3 are presented. The decay kinetics of the tyrosyl residue are complex for each compound. By use of a linked-function analysis, the fluorescence kinetics can be explained by a ground-state rotamer model. The linked function assumes that the preexponential weighting factors (amplitudes) of the fluorescence decay constants have the same relative relationship as the 1H NMR determined phenol side-chain rotamer populations. According to this model, the static quenching of the oxytocin fluorescence can be attributed to an interaction between one specific rotamer population of the tyrosine ring and the internal disulfide bridge.

Conformational studies on [Pro3, Gly4]-oxytocin in dimethyl sulfoxide by 1H nuclear magnetic resonance spectroscopy: evidence for a type II beta turn in the cyclic moiety.

A model for oxytocin has been previously proposed in which residues 3 and 4 occupy the corner positions in a beta turn (Urry, D. W., & Walter, R (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 956). The analogue [Pro(3),Gly(4)]-oxytocin (PGO) was used in proton magnetic resonance (1H NMR) studies designed to probe the contribution of these corner positions in forming a beta turn. Comparison of various 1H NMR parameters obtained at 220 MHz for backbone amide protons of PGO with those for the corresponding protons of oxytocin suggests that, to a first approximation, these two peptides may have similar backbone conformations in )CD3)2SO. Theoretically, the L-Pro lead toGly sequence in positions 3 and 4 of PGO would allow the formation of either a type I or type II beta turn. The two coupling constants between vicinal amide and C alpha protons for Gly(4) of PGO in (CD3)2SO are compatible with a type II beta turn in which 04, the dihedral angle about the bond between the backbone C alpha and N' atoms of Gly4, is appromximately +92 degrees, but not with a type I beta turn. A survey of peptides studied in other laboratories by X-ray and/or 1H NMR with reported type II beta turns with L-Pro lead toGly or Gly lead to Gly sequences in the corner positions revealed a close correlation between chemical shifts and vicinal coupling constants for the glycl residue in the second corner position. It is suggested that this criterion can form an additional basis for the characterization of beta turns. More studies are needed to determine the particular type of beta turn manifest in the cyclic moiety of oxytocin per se, although a reasonable working hypothesis is that oxytocin, similar to PGO, has a type II beta turn.

Spin-lattice relaxation times for 13C in isotope-enriched glycine accumulated in frog muscle.

Spin-lattice relaxation times (T1's) of 13C-enriched glycine accumulated in frog muscles were determined at 1 degrees C by the inversion-recovery (180 degrees -tau-90 degree pulse sequence) method and compared with the values obtained in free solution. The value of T1 for the alpha-13C nucleus of glycine in the tissue was 50% of that obtained in free solution. The observed value for T1 in the tissue was not concentration-dependent, and no difference in chemical shift was observed between tissue and free solution. Quantification of the area under the glycine peak suggested that the observed signal represents at least 80% of the intracellular glycine. An average nuclear Overhauser enhancement of 2.83 for intracellular glycine indicates that the relaxation mechanism within the cell is predominantly dipolar, as in free solution. The value of T1 for the 13C' nucleus of glycine in the tissue was 67% of that in a solution of similar concentration. A quantitative analysis of the findings suggests that the observed difference in the value of T1 between tissue and free solution results from a difference in viscosity. The data provide no evidence either for special organization of intracellular water or for glycine binding. It is proposed that intracellular diffusion coefficients may be determined from measurements of 13C T1's of 13C-enriched intracellular solutes.

Studies of individual amino acid residues of the decapeptide tyrocidine A by proton double-resonance difference spectroscopy in the correlation mode.

The cyclic decapeptide antibiotic tyrocidine A was studied by two relatively new methods, viz., correlation proton magnetic resonance (pmr) spectroscopy and double-resonance difference pmr spectroscopy. The correlation method of spectral accumulation provided pmr spectra of good resolution, and in addition the signal-to-noise ratio achieved per unit time of accumulation was much higher than that achieved by use of the conventional continuous wave (cw) method. Furthermore, when protonated solvents are used, the correlation mode of accumulation has a distinct advantage over pulse and fast Fourier transform (fft) methods currently in use. Double-resonance difference (drd) spectra of individual amino acid residues in tyrocidine A were obtained by the correlation method when the decoupling frequency was maintained at the center frequency of the appropriate C-alpha proton multiplet and at a level of power that totally decoupled vicinal C-alpha and C-beta protons; the resolution of these spectra was good, and the signal-to-noise ratio was high. The distinct patterns and spectral positions of the drd spectra were characteristic of the particular type of amino acid residue and, therefore, could be used as the basis for making assignments. Furthermore, the drd spectra revealed the spectral positions of individual C-alpha and C-beta proton transitions and therefore, upon spectral analysis, could provide the chemical shifts and coupling constants of these protons. Positions of transitions were revealed even though they were hidden by overlap in the corresponding conventional single- or double-resonance spectra.