Interaction of parvalbumin of pike II with calcium and terbium ions.

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from Förster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.

Towards assignment of secondary structures by anti-peptide antibodies. Specificity of the immune response to a beta-turn.

In an attempt to assign secondary structure elements to protein primary structures with antibodies, we synthesized a model peptide (beta-peptide: TVTVTDPGQTVTY) with a putative beta-turn structure and analysed the anti-peptide antibodies for their specificity towards the turn sequence. At least 50% of the peptide fraction adopts the intended conformation of a beta-turn (DPGQ) inserted between the two segments of an antiparallel beta-sheet structure. The specific anti-beta-peptide antibodies of the hyperimmune response bind the beta-turn containing epitope of the immunogenic beta-peptide with a three orders of magnitude higher affinity than the synthetic control peptide (Gly-peptide: GGGGGDPGQGGGG). The affinity of the antibodies with specificity for the beta-turn region increases from the primary to the hyperimmune response. Therefore, probing of secondary structure elements, i.e., of individual beta-turn regions, by anti-peptide antibodies now seems feasible for proteins of known sequence and may result in sequence assignments of secondary structures.

A solution equivalent of the 2Zn----4Zn transformation of insulin in the crystal.

Circular dichroic spectroscopy clearly reveals a solvent-induced conformational change of insulin in the presence of zinc ions. The spectral change corresponds to an increase in helix content. The transition observed in solution is an equivalent of the 2Zn----4Zn insulin transformation in the crystal. This is inferred from a series of observations. (1) The spectral effects are compatible with the refolding of the B-chain N-terminus into a helix known from crystal studies. (2) The spectral effects are induced by the very same conditions which are known to induce the 2Zn----4Zn insulin transformation in the crystal (i.e. threshold concentrations of NaCl, KSCN, NaI, for example). (3) They fail to be induced by the same conditions that fail to induce the crystal transformation (e.g. Ni2+ instead of Zn2+). It is concluded that the potential to undergo the transition resides in the hexamer since neither insulin dimers nor monomeric des-pentapeptideB26-30-insulin respond detectably to high halide concentration. Secondly the ability of zinc ions to accommodate tetrahedral coordination allows the transition which is not permitted by other divalent metal ions. Thirdly the transition is independent of the off-axial tetrahedral zinc coordination sites since it occurs in [AlaB5]insulin which lacks the B5 histidine necessary for their formation. A symmetrically rearranged hexamer thus appears possible with two tetrahedrally coordinated zinc ions on the threefold axis; this is consistent with the observation that in native insulin two zinc ions per hexamer are sufficient to produce the full spectral effect. The amount of additional helix derived from the circular dichroic spectral change, however, cannot settle whether the transition comprises only three or all six of the subunits to yield a symmetrical hexamer. Finally the transformation in solution evidently still occurs in an intramolecularly A1-B29-cross-linked insulin in spite of the partially reduced flexibility.

Two mutant forms of human insulin. Structural consequences of the substitution of invariant B24- or B25-phenylalanine by leucine.

In 1979 the first abnormal human insulin was discovered. With the minute samples from the patient a Phe leads to Leu replacement could be established in either position B24 or B25. For the unequivocal localization of the substitution both the Leu analogues had to be prepared by semisynthesis. While another laboratory did this with the sequence of porcine insulin, here we are dealing with the true analogues of human insulin. In the present paper the structural consequences of the substitutions are investigated. Human insulin obtained by total synthesis served as a reference. Its CD spectral properties are herewith documented. According to the substantial deviations of the CD spectrum of [Leu B24]insulin, the introduction of the new side-chain forces conformational changes to occur not only in its immediate surrounding but also in the peptide chain. The failure to give the typical CD spectral response to variations of protein and zinc concentration indicates that the ability to form quaternary structure is impaired. Though dimerization was confirmed by gel chromatography to be largely reduced, it is concluded that, in addition, interactions normally responsible for the increase in tyrosine-CD with association are weakened. [Leu B25]insulin, on the other hand, does exhibit all CD spectral effects characteristic of the native hormone, though quantitatively somewhat reduced. The CD spectroscopic results are in full agreement with the computergraphic analysis of the sterical consequences of the substitutions. For B24-leucine an acceptable packing without movements of the mainchain and/or B15-leucine and without affecting dimerization is impossible, whereas B25-leucine can be accommodated without causing bad contacts either in the monomer or in the dimer. The structural results do not explain why [Leu B25]-insulin should have a lower biological activity than the B24 analogue, 2.1 +/- 0.3% versus 20.9 +/- 2.8%, in the fat cell test. They suggest, however, an important but not critical stereospecific role for the B25-phenylalanine in binding.