Robert J. Anderegg

Activation of a Novel Calcium-dependent Protein-tyrosine Kinase CORRELATION WITH c-Jun N-TERMINAL KINASE BUT NOT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cβ, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ∼ 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFα) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the other calcium-dependent.

Specific and nonspecific dimer formation in the electrospray ionization mass spectrometry of oligonucleotides.

Specific and nonspecific noncovalent dimer ions of oligonucleotides (ODNs) were observed when mixtures of complementary or noncomplementary strands were analyzed via negative ion electrospray ionization mass spectrometry. Dimer formation was concentration dependent and nearly always occurred when the concentration of ODN exceeded 100 µM. Dimers were observed even for short-length ODNs for which the melting temperature (Tm) was well below the experimental temperature and which, therefore, would not be expected to form stable solution duplexes. The abundance of the heterodimer ions seems to correlate with the number of expected hydrogen bonds from Watson-Crick base pairing. As the energy of the incoming ion beam (orifice potential) was increased, the absolute and relative abundance of the dimer ions unexpectedly increased.

The mass spectrometry of helical unfolding in peptides

Two model peptides, melittin and a growth hormone releasing factor (GRF) analog, have been studied by mass spectrometry and tandem mass spectrometry during the course of their deuterium exchange. Both peptides are known from previous work to form α-helices in solution. When the peptides are exposed to deuterated solvents, their masses increase as deuterium atoms replace protons in the exchangeable sites of the peptides. The mass spectrometry results clearly indicate multiple populations of exchangeable protons: Some exchange very fast, and are presumably on the surface and not involved in hydrogen bonding; others exchange much more slowly, indicating that they are probably participating in hydrogen bonding.Tandem mass spectrometric experiments were conducted, and the masses of the product (fragment) ions were used to determine where in the peptide the deuterium atoms were incorporated. The results agree very well with NMR studies of the same peptides. Melittin appears as two helical segments with a kink around Pro-14. The GRF analog contains a single long helix, spanning almost the entire length of the peptide. The dynamics of the unfolding of the helices can also be explored by observing how the exchange progresses with time.

Analytical supercritical fluid chromatography using fully automated column and modifier selection valves for the rapid development of chiral separations

Automated analytical supercritical fluid chromatography is used to separate enantiomers of pharmaceutical compounds in the drug discovery laboratory. Modification of a commercial instrument to incorporate a six-way column selection valve, multiple chiral columns based on derivatized cellulose or amylose, and a four-way modifier selection valve provides a powerful combination for the rapid development of chiral separations. A wide set of columns and conditions can be tested sequentially, including unattended operation overnight. This paper shows that similar racemic compounds, even those from the same molecular class, are separated using different column and modifier combinations. Therefore, the use of program-controlled column and modifier selectors has great advantages. Using the fully automated system, the optimal chiral separation of several compounds can be obtained unattended within 24 h.

Probing the helical content of growth hormone-releasing factor analogs using electrospray ionization mass spectrometry

A series of growth hormone-releasing factor analogs have been studied by both circular dichroism and electrospray ionization mass spectrometry (ESI/MS). The peptides are 32 residues long and are known to adopt a random-coil structure in aqueous solution but become increasingly helical as the proportion of organic solvent is increased. Deuterium exchange was observed as an increase in mass of the peptide, as measured by ESI/MS. Rates of exchange were measured and half-lives calculated for analogs containing amino acid substitutions designed to promote or discourage helix formation. Exchange was slower in peptides that are helical (as shown by circular dichroism) than in randomly coiled peptides. Solution conditions that favor helix formation also produced slower exchange rates. These studies suggest that ESI/MS can provide date about the extent and stability of helix formation.

Characterization of femtomole levels of proteins in solution using rapid proteolysis and nanoelectrospray ionization mass spectrometry

A method for the rapid proteolytic digestion of low picomole to low femtomole amounts of proteins in solution using a capillary immobilized protease column is presented. Dilute protein samples are passed through a “μ-digestion” column packed with Poroszyme™ immobilized trypsin for generation of proteolytic fragments in less than 10 min. After digestion, nanoelectrospray ionization mass spectrometry (NanoES) is used to generate a peptide map, and peptides of interest are subjected to MS/MS from the same sample. By digesting only 100 fmol of the protein src kinase and 30 fmol of the protein lck kinase with a tryptic μ-digestion column, we obtained sufficient data from NanoES-MS and MS/MS to unambiguously identify both proteins using database searching. This approach was also used to locate a phosphorylation site on lck kinase starting with only 300 fmol of protein. The method was successfully used to identify an E. coli cold shock protein in a fraction collected from a two-dimensional HPLC separation of an E. coli cell lysate. The μ-digestion column was found to be less susceptible to adsorptive losses than solution digests, thus allowing for digestion and enhanced recovery of peptides from even low fmol amounts of protein in solution.

Iterative size-exclusion chromatography coupled with liquid chromatographic mass spectrometry to enrich and identify tight-binding ligands from complex mixtures

Abstract A method is described for the enrichment of tight-binding ligands from complex mixtures. The mixture is equilibrated with a protein or receptor, and protein: ligand complexes are separated from unbound ligands by size-exclusion. If the resulting mixture is allowed to re-establish its equilibrium and then passed again through the size-exclusion column, significant enrichment of tight-binding ligands is realized, even in cases where the weak-binders are present in large excess. Ligands are identified by liquid chromatography and mass spectrometry following the size-exclusion step. The process can be repeated multiple times to further enrich ligands. Analysis of pools of ligands generated by combinatorial chemistry is an obvious application of the technique.

A Multidimensional Approach to Protein Characterization

When mass spectrometry (MS) is used to study protein primary structure, it is used in a “static” mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the “dimensionality” of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.

Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a β structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12–Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12–Ser13 peptide bond may be due to its decreased accessibility caused by Cu2+-induced folding of betabellin 15D.

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu2+. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu2+ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu2+ but not in its absence. The observed width of the fibrils (7 ± 2 nm) was consistent with the width (6.8 nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.