SynopsisThe 'H-nmr chemical shifts and the spin-spin coupling constants of the common amino acid residues were measured in solutions of the linear tetrapeptides H-Gly-Gly-X-L-Ala-OH in D2O and H20, and the influence of X on the nmr parameters of the neighboring residues Gly 2 and Ala 4 was investigated. The titration parameters for the side chains of Asp, Glu, Lys, Tyr, and His were determined. The pK, values obtained in D20, with the use of pHmeter readings with a combination glass electrode uncorrected for istope effects, were 0.06 pH units higher in the acidic range and 0.10 pH units higher in the basic range than the corresponding pK, values in HzO. This suggests that the present data are suitable "random-coil" 'H-nmr parameters for conformational studies of polypeptide chains in D2O and H20 solutions.

This paper shows that backbone amide proton titration shifts in polypeptide chains are a very sensitive manifestation of intramolecular hydrogen bonding between carboxylate groups and backbone amide protons. The population of specific hydrogen‐bonded structures in the ensemble of species that constitutes the conformation of a flexible nonglobular linear peptide can be determined from the extent of the titration shifts. As an illustration, an investigation of the molecular conformation of the linear peptide H‐Gly‐Gly‐L‐Glu‐L‐Ala‐OH is described. The proposed use of amide proton titration shifts for investigating polypeptide conformation is based on 360‐MHz 1H‐nmr studies of selected linear oligopeptides in H2O solutions. It was found that only a very limited number of amide protons in a polypeptide chain show sizable intrinsic intration shifts arising from through‐bond interactions with ionizable groups. These are the amide proton of the C‐terminal amino acid residue, the amide protons of Asp and the residues following Asp, and possibly the amide proton of the residue next to the N‐terminus. Since the intrinsic titration shifts are upfield, the downfield titration shifts arising from conformation‐dependent through‐space interactions, in particular hydrogen bonding between the amide protons and carboxylate groups, can readily be identified.

Dilute aqueous solutions of glucagon were investigated by high-resolution 'H nuclear magnetic resonance at 360 MHz. Monomeric glucagon was found to adopt predominantly an extended flexible conformation which contains, however, a local non-random spatial structure involving the fragment -Phe-22 -Val-23 -Gln-24 -Trp-25 -. This local conformation is preserved in the partial sequence 22 -26 and could thus be characterized in detail. Two interesting conclusions resulted from these experiments. One is that the local spatial structure in the fragment 22-25 of glucagon is identical to that observed in the fragment 20 -23 of the human parathyroid hormone. Secondly, the backbone conformation in the C-terminal fragment of glucagon in solution must be different from the a-helical structure observed in single crystals of glucagon. These new structural data are analyzed with regard to relationships with glucagon binding to the target cells.Glucagon is a polypeptide hormone which consists of one linear peptide chain with 29 amino acid residues [I]. The biological function of glucagon was found to be related with specific binding to a plasma membrane receptor, which results in stimulation of adenylate cyclase [2]. To gain more detailed insight into the structurc-function relations, the molecular conformation of glucagon was extensively investigated, both in single crystals and in solution. In the single crystal X-ray structure, glucagon trimers were observed, where the individual molecules adopt a mainly a-helical conformation [3]. Glucagon in solution was investigated by various spectroscopic techniques, such as circular dichroism [4 -71, optically detected magnetic resonance [8] and nuclear magnetic resonance (NMR) [9-111. Two observations stand out among the results of the solution studies. One is that the conformational properties of glucagon manifested in the spectral parameters depend strongly on the solution conditions. Thus it was reported that glucagon in freshly prepared dilute aqueous solution adopts predominantly a flexible 'random coil' form, while under different solution conditions or simply after prolonged standing of the solutions more highly structured aggregates were observed [4 -81. Conformational changes are apparently also induced by interactions Abbreviaticms. NMR, nuclear magnetic resonancc; Z, benzyloxycarbonyl protecting group. of glucagon with lipids or detergents [12,13]. Secondly, the descriptions of the solution conformations are throughout in the terminology of circular dichroism measurements, i.e. random coil structures and those with varying a-helix of 8-pleated sheet contents were distinguished [4-91.In view of the pronounced conformational polymorphism shown by numerous investigations under different experimental conditions, it appears particularly important to complement the single-crystal X-ray structure analysis by detailed conformational studies in well-defined solutions. High-resolution NMR is the method of choice for obtaining a niany-parameter characterization of the solution conformati...

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Previous studies had shown that the molecular conformation of the synthetic human parathyroid hormone fragment 1 -34 in dilute aqueous solution contained a local non-random structure formed by the four consecutive residues -Val-21 -Gln-22 -Trp-23 -Leu-24 -. This paper gives a detailed description of this local spatial structure obtained from high resolution 'H NMR studies at 360 MHz of several peptide analogs of the partial sequence 20-24. The most important spectral parameters were high-field shifts of the a and y protons of Val-21, the spin-spin coupling constants related to the rotamer populations of the side-chains of Val-21 and Trp-23, and pH titration shifts of the amide proton resonances. It was found that the backbone fragment 20-24 is so arranged that the side-chain of Val-21 is located next to the indole ring plane of Trp-23; evidence is presented that this non-random structure is mainly stabilized by hydrophobic interactions between the side-chains of Val-21 and Trp-23. The thermal population of the observed molecular structure at room temperature was estimated from the nuclear magnetic resonance data to be approximately 20%.In a previous paper it was shown that the synthetic human parathyroid hormone fragment 1 -34 exists in aqueous solution predominantly in a flexible extended form, where, however, a local non-random structure was also clearly manifested in the ' H nuclear magnetic resonance (NMR) parameters [l]. This non-random conformation was then found to involve the amino acid residues -Val-21 -Gln-22-Trp-23 -Leu-24 -, and it was demonstrated that the same structure was preserved in the pentapeptide Arg-Val-Gln-Trp-Leu[l]. Our interest in this structure was further enhanced, since the tetrapeptide sequence 21 -24 in the parathyroid hormone corresponds to the tetrapeptide sequence 23 -26 in glucagon [2,3]. The present paper describes a detailed investigation of the spatial arrangement of the tetrapeptide segment 21 -24 in the parathyroid hormone 1 -34.The strategy used for this study was to compare the spatial structures of different synthetic peptide analogs by high-resolution 'H NMR techniques. On the basis of the requirements of compatibility with all the NMR data and with the criteria of closest interAbhreviations. NMR, nuclear magnetic resonance; parathyroid hormone 1 -34, synthetic N-terminal fragment 1 -34 of the human parathyroid hormone. atomic distances, a molecular model for the tripeptide segment -Val-21 -Gln-22 -Trp-23 -in the parathyroid hormone 1 -34 was constructed. With the use of the atomic coordinates obtained from this model, the thermal population of the local spatial structure was estimated in a quantitative analysis of the NMR data. MATERIALS AND METHODS Peptidr SynthesisThe synthesis of parathyroid hormone 1-34 according to the sequence determined by Brewer et al. [4] and of several partial sequences thereof was described previously [ 5 ] . This concerns in particular the partial sequences Met-1