While electrospray (ESI) mass spectrometry has already established its potential for the characterization of non‐covalent protein complexes, matrix‐assisted laser desorption/ionization mass spectrometry (MALDI/MS) seemed not to be applicable hitherto because of limitations in matrix chemistry and sample preparation. In this work, a sample preparation method has been developed in which 6‐aza‐2‐thiothymine (ATT) was used as a matrix without any addition of organic cosolvents, and proteins were dissolved in aqueous buffers such as ammonium hydrogencarbonate, ammonium citrate and ammonium acetate. Under these conditions, the intact non‐covalent protein complexes, RNAse S, the non‐covalent complex of S‐protein and S‐peptide and specific dimers of coiled‐coil leucine zipper polypeptides were observed by UV‐MALDI/MS. The specificity of complex formation was ascertained by admixture of non‐specific peptides which did not yield detectable aggregate ions. In addition, on‐target tryptic digestion of cytochrome c and leucine zipper peptides was carried out after MALDI/MS molecular mass determination in the presence of the ATT matrix. Mass spectrometric analyses of these tryptic digests yielded spectra that showed complete digestion of the proteins. These results indicate that proteins maintained intact tertiary structures necessary for the formation of specific non‐covalent complexes, and that trypsin retained its functional enzymatic structure and full biological activity with the present sample preparation method.
The cop operons of Helicobacter pylori andHelicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon ofEnterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.
An efficient synthesis for human-identical lung surfactant protein SP-C is described with a semi-automated solid phase synthesizer using Fmoc chemistry. Double coupling and acetic anhydride capping procedures were employed for synthetic cycles within the highly hydrophobic C-terminal domain of SP-C. Isolation of the protein was performed by mild cleavage and deprotection conditions and subsequent HPLC purification yielding a highly homogeneous protein as established by sequence determination, electrospray, plasma desorption and MALDI mass spectrometry. A general method has been employed for the preparation of Cys-palmitoylated protein by using temporary Cys(tButhio) protection, in situ deprotection with beta-mercaptoethanol and selective palmitoylation of resin-bound SP-C. The mild synthesis and isolation conditions provide SP-C with a high alpha-helical content, comparable to that of the natural SP-C, as assessed by CD spectra. Furthermore, first biophysical data indicate a surfactant activity comparable to that of the natural protein.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.