Gln Side Chain Research Articles

Nuclear magnetic resonance conformational studies of the Cα–Cβfragments of oxytocin, oxytocinoic acid, and tocinoic acid in aqueous solution

We have recorded the 1H n.m.r. spectra (300 and 270 MHz) of oxytocin, oxytocinoic acid, and tocinoic acid in aqueous solution in the presence and absence of lanthanide ions. From the analysis of the CαHCβH2 regions of the spectra, the Jα,β coupling constants have been derived and found to be similar for corresponding residues in the different molecules indicating these fragments to have similar conformations in the three molecules. The coupling constants for the side-chains of Tyr, Ile, Gln, and Asn have been used to estimate the fractional populations of the three minimum-energy staggered rotamers. The Cys(1)–Cys(6) fragments also have similar conformations and, for tocinoic acid, this conformation has been partially defined by analysing the combined coupling constant and lanthanide induced shift data. The binding of lanthanide ions does not affect the conformation (no change in coupling constants when La3+ ions bind) and because tocinoic acid, oxytocin, and oxytocinoic acid have similar conformations for their Cys(1)–Cys(6) fragments, the conformational information derived for tocinoic acid will be valid also for oxytocin and oxytocinoic acid.

The conformation of oxytocin in dimethylsulfoxide as revealed by carbon-13 spin-lattice relaxation times

Information was obtained on rates of overall molecular reorientation and segmental motion of amino acid sidechains of oxytocin in dimethylsulfoxide by determination of spin-lattice relaxation times (T 1) at 25 MHz for carbon-13 in natural abundance in the hormone. The T 1 values of the α-carbons of amino acid residues located in the 20-membered ring of oxytocin are all about 50 msec. The overall correlation time for the hormone backbone was estimated to be 8.8 × 10 −10 sec. The sidechains of Tyr, Ile and Gln undergo segmental motion with respect to the backbone of the ring. The T 1 value of the α-carbon of the Leu residue is greater than for any α-carbon in the ring, indicating an increased mobility of the backbone of the C-terminal acyclic peptide as compared to the ring. The β- and γ-carbons of the Pro residue undergo an exo-endo interconversion with regard to the plane formed by α-carbon, δ-carbon and N atom of the Pro pyrollidine ring. These data are discussed in light of results from other experimental and theoretical studies, including carbon-13 spin-lattice relaxation times for oxytocin in aqueous solution.

Conformational energy studies of oxytocin and its cyclic moiety.

Conformational energies were calculated for oxytocin in water, starting with a conformation proposed from nuclear magnetic resonance measurements in [U-(2)H](CH(3))(2)SO. Calculations on the isolated ring showed that conformations with one transannular hydrogen bond had the same energies as those without such bonds; those with two such hydrogen bonds do not appear to form. Calculations on the whole molecule also indicated the existence of several low-energy minima in the energy surface, and no preference for hydrogen-bond formation in the cyclic moiety; the hydrogen bond proposed between the Gly peptide NH and the Cys-6 C=O in the acyclic moiety can form. The proposed proximity of the tail to the ring is one of two low-energy conformations found. The Tyr side chain had two conformations of comparable energy, one over the ring between the Gln and Asn side chains, and the other with the Tyr side chain away from the ring. The oxytocin molecule appears to be flexible, and is probably sensitive to changes in its environment.

Conformational changes accompanying the formation of chymotrypsin-substrate complexes. Evidence for the involvement of an N-terminal α-amino group in the activity and the conformation of the enzyme

Crystals of γ-chymotrypsin (γ-CHT) grown at pH 7.0 are stable from pH 2.0 to 11.0. Crystalline γ-CHT therefore provides an unusually favourable system to observe the structure of a protein and its bound solvent over a broad range of pH. In this report we describe the high-resolution refined structure of γ-CHT at pH values of 2.0, 7.0 and 10.5. The apparent tetrapeptide seen bound in the active site of γ-CHT at pH 7.0 is also present at pH 2.0 and 10.5 although it is better defined at low pH. A comparison of the respective structures shows that there is additional electron density in the low pH structure at the point where the side-chain of Ser 195 approaches most closely to the presumptive inhibitor. This suggests that the adduct is most likely to be covalently linked to the enzyme at low pH and to be non-covalent at higher pH. As the pH is lowered from 7.0 to 2.0, the side-chain of His 40 rotates approximately 120° about its CαCβ bond and, in concert, the side-chain of Gln 34 also rotates approximately 140° about its CαCβ bond. Apart from these localized rearrangements in the vicinity of His 40, the structure of γ-CHT at pH 2.0 is very similar to that at neutral pH. The structure of γ-CHT at pH 10.5 is also seen to be almost identical with that at neutral pH. There is no indication that the internal salt bridge between Asp 194 and the α-amino group of lle 16 begins to dissociate at pH 10.5. With the exception of the vicinity of His 40, the structure of the bound solvent in the crystal structures at low, neutral and high pH is very similar. There is no clearcut example where the solvent is seen to rearrange in response to the titration of a group on the surface of the protein.