Identifying structure-function relationships in four-helix bundle cytokines: towards de novo mimetics design.

Many cytokines (growth-factor proteins) are constructed from a common four-helix bundle structural framework. Rapid advances have been made in relating the structure and function of a growing number of four-helix bundle cytokines. This understanding opens the way to design de novo mimetics through such strategies as cytokine hybrids, structure-excerpted scaffolds and contact residue topology mimics. These may provide leads for agonists and antagonists of cell growth and differentiation.

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis.

The potential of the common biosynthetic precursor of neurophysin and neuropeptide hormones to self-associate has been assessed by quantitative affinity chromatographic analysis. The precursor form, with the hormone sequence in the amino terminal region and assumed able to interact intramolecularly with the hormone binding site of the neurophysin domain of the folded precursor, exhibits an affinity for neurophysin-agarose which is intermediate between those of unliganded neurophysin and non-covalently hormone-liganded neurophysin. The results lead to a prediction that neurophysin self-association is established upon precursor synthesis and prior to limited proteolysis of the precursor to release mature neurophysin and hormone components. Such self-association could play a role in packaging of the precursor into secretory granules and in regulating subsequent precursor processing events within the granules.

Isolation and sequence analysis of oxytocin from the sheep corpus luteum.

Studies have revealed that the corpus luteum (CL) of the sheep releases oxytocin (OT) -like immunoreactivity under normal and physiological conditions. We have now purified and completely characterized the OT-like species from ovine CL and established by Edman degradation and comparative reverse-phase HPLC its identity with hypothalamic oxytocin. On the basis of radioimmunoassay, the characterized oxytocin was the only peptide possessing OT-like immunoreactivity. This study represents the first identification by sequence analysis of oxytocin outside the central nervous system.

Kinetic analysis of a protein antigen-antibody interaction limited by mass transport on an optical biosensor.

Using BIAcore technology, we determined the rate constants for a protein antigen-antibody interaction that was mass transport limited on the optical biosensor. The antigen consisted of a soluble form of the human T-cell receptor CD4 (two amino terminal domains, D1D2) and the antibody was an anti-CD4 monoclonal from monkey engineered with the constant domains from human IgG1. High quality response data were obtained for this interaction by orienting the attachment of the antibody on the sensor surface and correcting for instrument artifacts with control experiments. Using numerical integration and global fitting, we demonstrate that a mass transport limited reaction was the only model of those tested that described well D1D2 binding to three different surface densities of the antibody. Statistical profiling techniques showed that the error space and correlation for the parameters in the non-linear model were essentially linear, but only when the model was simultaneously fitted to data from multiple surface densities. The "on" and "off" rate constants (1.2 x 10(-6) M-1 s-1 and 2.9 x 10(-4) s-1) determined from the kinetic analysis predict an equilibrium dissociation constant (KD = 0.24 +/- 0.01 nM) that agrees with the value measured in solution by titration calorimetry (KD = 0.2 +/- 0.1 nM). The results indicate that, although the D1D2-antibody reaction is partially controlled by mass transport on the optical biosensor, by optimizing the experimental design and analyzing data from multiple surface densities it is possible to determine accurate estimates of the intrinsic equilibrium and kinetic rate constants.

Molecular diagnostics using analytical immuno high performance liquid affinity chromatography.

Analytical immuno high performance liquid affinity chromatography (analytical immuno HPLAC) was evaluated as a molecular diagnostic tool. Antibodies raised in rabbits against bovine neurophysin II were immobilized through Protein A crosslinking onto coated silica. Interaction of immobilized antibody with mobile antigen was characterized by zonal and frontal elutions of 14C-labeled bovine neurophysin II under isocratic, nondenaturing conditions. The chromatographic behavior shows that analytical immuno HPLAC with immobilized antibodies can be used to detect the number and functional nature of matrix-interacting antigens in mixtures, thus providing a quantitative chromatographic technology for "antigen mapping."

Use of enzymes in peptide synthesis.

Recent experiments in several laboratories have emphasized the benefits of proteolytic enzymes as effective catalysts for the formation of peptide bonds for synthesis and semisynthesis. This review summarizes successful applications in both stepwise synthesis for small peptides and fragment condensation to produce large polypeptides and proteins.

Analytical affinity chromatography in studies of molecular recognition in biology: a review.

Measuring macromolecular and cellular interactions remains central to the study of recognition in biology and its application in biotechnology. Analytical affinity chromatography provides a versatile methodology to detect and quantitate such interactions. Both zonal and frontal elution approaches have been developed, essentially in parallel, for analytical affinity chromatography. A close quantitative relatedness of chromatographically obtained equilibrium constants and analogous constants determined fully in solution has been found for a growing number of proteins. This consistently observed correlation has formed the basis for extending theoretical treatments in order to evaluate not only monovalent molecular systems of varying types but also multivalently interacting macromolecules, including those which exhibit cooperativity. The potential to measure chemical rate constants by affinity chromatography also has been recognized, and experimental tests of the available theory are being made. As a micromethod, the quantitative use of affinity chromatography has important applicability for biochemical analysis of an increasing array of biologically active molecules being discovered and isolated but available in only relatively small amounts. Analytical affinity chromatography thus provides a means to use matrix--mobile interactant systems to study mechanisms of biomolecular interactions and therein to attain an understanding of such interactions which often is not easily achieved by solution methods alone.

Bioaffinity chromatography: synergy between interactive chromatography and molecular recognition for the separation and analysis of macromolecules.

Affinity chromatography, commonly regarded as an integral tool in macromolecular separation sciences, also provides an analytical method to study structure-function relationships of macromolecular interaction processes and to design recognition molecules. The latter, as found recently for the case of antisense peptides, may be useful as affinity agents in immobilized forms to effect new types of biomolecular separation.

Semisynthetic peptides and proteins.

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Characterization of the size and 5' cap of messenger RNA encoding neurophysin precursors.

The coding activity of bovine hypothalamic poly A+ mRNA for neurophysin I and II immunoreactive proteins was characterized with respect to size and 5' cap. The mRNA was fractionated by methylmercuric hydroxide agarose gel electrophoresis and subsequently translated in vitro in rabbit reticulocyte lysates. Alternatively, mRNA was fractionated by gel exclusion HPLC and translated in wheat germ extracts. Immunoprecipitated translation products were analyzed by gel exclusion HPLC. Neurophysin-immunospecific protein of approximately 17,000 daltons, the size expected for the neuropeptide hormone-neurophysin precursors, was encoded by mRNA species of two size classes. The smaller class of mRNA's was of the size expected from the size of the precursor proteins. The larger class was 5-10 times larger. The low K+ concentration optimum for translation of unfractionated mRNA encoding neurophysin I immunoreactive proteins and the inability of a cap analogue to inhibit this translation suggest that mRNA species encoding neurophysin I-immunoreactive translation products are incompletely capped. By contrast, the mRNA encoding neurophysin II-immunoreactive products appear to contain a normal cap structure.

Structural requirements of peptide hormone binding for peptide-potentiated self-association of bovine neurophysin II.

Site-specific, truncated, and sequence-simplified analogs of the hormone [Arg8]vasopressin were investigated for the relationship between their abilities to recognize immobilized bovine neurophysin and to promote neurophysin self-association. Peptide binding to neurophysin was measured quantitatively by analytical high performance affinity chromatography on immobilized bovine neurophysin II. Neurophysin self-association, measured as binding of soluble to immobilized neurophysin, was promoted (made higher affinity) by soluble peptide hormone and its analogs, with the effect of particular peptides being proportional to their binding affinities for neurophysin. Sequence-redesigned peptides able to recognize neurophysin, including dipeptide amides, were able to potentiate the self-association to the same extent as the natural hormone when tested at concentrations adjusted to effect equal degrees of saturation of neurophysin. The relationship between peptide affinity to neurophysin and the potentiation of self-association suggests that the latter is directly dependent on the former and can occur even with limited segments of hormone sequence. The data fit best to a model in which hormone binding and self-association surfaces of neurophysin are separate and linked through the neurophysin molecule to produce cooperativity (hormone-promoted self-association). Given that only limited structural elements of hormone are required for promoting self-association, the results fit less well with models in which cooperativity requires that hormone make dimer-stabilizing contacts with both self-associating subunits of neurophysin simultaneously.

Pituitary enzyme conversion of putative synthetic oxytocin precursor intermediates.

Neurosecretory granule lysate from bovine posterior pituitary was shown to contain both carboxypeptidase B and amidating activities. The former sequentially releases COOH-terminal basic residues from the oxytocin biosynthetic precursor fragment oxytocinyl-GKR (CYIQNCPLGKR) to form oxytocinyl-GK and then oxytocinyl-G. The amidating enzyme converts the resulting oxytocinyl-G into oxytocin (CYIQNCPLG-NH2). The carboxypeptidase B was separated from a less specific carboxypeptidase present in granule lysate by gel filtration on Sephacryl S-300. Percoll density gradient centrifugation (after preliminary differential centrifugation) also yielded granule fractions enriched in the specific carboxypeptidase B activity. The carboxypeptidase B which converts the oxytocinyl peptides showed a fairly sharp pH dependence with an optimum of 5.5-6, was activated by cobalt ion, and was inhibited by cupric ion, EDTA, and a thiol protease inhibitor, p-chloromercuribenzoate. The amidating activity which converts oxytocinyl-G to oxytocin was competed by degradation due to proteases and/or peptidases present in lysate of Percoll gradient-derived granules. Oxytocinyl-GKR was shown by analytical affinity chromatography to bind noncovalently to neurophysin with an affinity close to that of mature oxytocin. This binding activity and the observation of carboxypeptidase B activity in the presence of large concentrations of neurophysin are consistent with the view that the exoproteolytic processing and amidation steps which occur after initial endoproteolysis of pro-oxytocin/neurophysin likely take place on oxytocin intermediate peptides which are bound in noncovalent complexes with the neurophysin domain from the precursor.

Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor.

An oxytocin/bovine neurophysin I biosynthetic precursor, [N epsilon-diacetimidyl-30,71, des-His106]pro-OT/BNPI, was synthesized from a synthetic oxytocinyl peptide, 1/2Cys-Tyr-Ile-Gln-Asn-1/2Cys-Pro-Leu-Gly-Gly-Lys-Arg, and native neurophysin by chemical semisynthesis. The semisynthetic precursor contains the entire sequence of the biosynthetic precursor deduced from the complementary DNA structure except for omission of the carboxyl-terminal histidine residue. The covalent structure of the semisynthetic product was verified by amino acid analysis and amino-terminal analysis. Analytical affinity chromatography was employed to evaluate noncovalent binding properties of the precursor. The precursor does not bind significantly to immobilized Met-Tyr-Phe, a hormone binding site ligand. In contrast, the acetimidated precursor binds to immobilized bovine neurophysin II, with a 13-fold higher affinity than does acetimidated neurophysin itself. When a hormonal ligand, [Lys8]vasopressin, was added to the elution buffer at the concentration of 0.1 mM so that a major portion of the immobilized BNPII was liganded, the affinity between the immobilized liganded BNPII and the precursor was enhanced 8-fold and approached the affinity for the liganded (bovine neurophysin I-immobilized BNPII) interaction. The data imply that the precursor can self-associate and that this self-association is closely related to that of liganded neurophysin. The tripeptide affinity matrix data argue that, in the precursor, the ligand binding site of the neurophysin domain is occupied intramolecularly by the hormone domain. The data verify the view that both the self-association surface and hormone binding site are established upon precursor folding. A disulfide stability analysis showed the resistance, to disulfide interchange by dithiothreitol, of semisynthetic precursor but not of neurophysin, as judged by protein association and peptide ligand binding activities, respectively. The results argue that the molecular structure of the precursor is established upon precursor folding and before enzymatic processing that produces mature hormone and neurophysin.

Quantitative affinity high-performance liquid chromatography of neuroendocrine polypeptides using porous and non-porous glass derivatives.

Analytical affinity HPLC was developed to isolate and characterize neuroendocrine peptide/protein components. Bovine neurophysin II (NP-II) was covalently immobilized on succinamidopropyl derivatives of both controlled-pore glass (CPG) and non-porous glass (NPG). These derivatives were packed into 25 X 0.46 cm I.D. stainless-steel columns and incorporated into a high-performance liquid chromatograph. Interaction of [3H]Arg8-vasopressin ([3H]AVP) with NP-II was examined by chromatography of AVP on both CPG and NPG affinity matrices. Zonal elution profiles of [3H]AVP on NPG matrix showed, as predicted theoretically, a linear dependence of retardation on the concentration of hormone injected. The data permit calculation of the equilibrium dissociation constant for the NP-II/AVP interaction. Elution characteristics also were measured by frontal analysis of large-zone chromatography experiments, the results of which were in good agreement with the zonal elution analysis. Affinity resulting from dimerization also was studied by chromatography of [125I]NP-II on the NPG matrix. In this case, concentration dependence of retardation was non-linear, again as predicted theoretically. Off-rate kinetic constants for dissociation of the mobile interactant from the stationary phase also were obtained. The studies illustrate the utility of analytical affinity HPLC on non-porous beads for measuring relative affinities for various soluble ligands with small amounts of material. Chromatography on the CPG column proved useful for purification of microscale amounts of [3H]AVP.