Peptide mapping and disulfide bond analysis of the cytoplasmic region of an intrinsic membrane protein by mass spectrometry.

Intrinsic membrane proteins pose substantial obstacles to analysis by common analytical techniques due to their hydrophobic nature and solubilization requirements. This is the case for studies involving HPLC coupled to mass spectrometry. We have developed an HPLC/mass spectrometry approach to explore and map the peptide sequence of the SERCA1a Ca(2+)-ATPase from the sarcoplasmic reticulum an integral membrane protein of 110 kDa. After extensive proteolysis of the protein, the mass of the proteolytic fragments was analyzed by HPLC/mass spectrometry. Only part of the cytoplasmic fragments was recovered under nondenaturing conditions. On the other hand, peptide fragments obtained under denaturing conditions were found to cover nearly all the cytoplasmic region. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase contains 24 cysteine residues, 18 of which are in the cytosolic or lumenal region of the protein. Peptides containing free cysteines were identified by a mass increase resulting from carboxyamidomethylation of the cysteines with iodoacetamide. Alkylation reactions were executed either before or after reduction of the peptide fragments by dithiothreitol. Analysis of the mass of the fragments indicates that no disulfide bonds exist in the cytoplasmic portion of SR Ca(2+)-ATPase.

Concentration jump experiments for the precise determination of rate constants of reverse reactions in the millisecond time range.

Low dissociation or reverse rate constants of single-step or multistep complex formation equilibria are usually obtained with reduced precision from standard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jumps, based on two different step-motor-driven mixing setups, are performed with 60-300-fold dilutions that allow the precise, convenient, and independent determination of dissociation rate constants in the range of approximately 0.1-100 s(-1) in a single stopped-flow dissociation experiment. A theoretical basis is developed for the design and for the evaluation of such dilution experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg2+ to 8-hydroxyquinoline as well as of Ca2+ and K+ to the cryptand [2.2.2], by carrying out standard stopped-flow binding as well as dissociation experiments employing various dilution factors. The advantage of the dilution method for investigating chemical and biological systems is emphasized.

Bimolecular DNA triplexes: duplex extensions show implications for H-form DNA stability.

H-form DNA has recently been shown to be biologically relevant by its involvement in the process of homologous recombination [Kohwi, Y. , and Panchenko, Y. (1993) Genes Dev. 7, 1766-1778]. A bimolecular DNA triple-stranded structure (triplex) is central to the formation of H-form DNA. Understanding the formation and factors governing the stability of such bimolecular triplexes is necessary to fully elucidate the structure/function relationship of H-form DNA. In this study, we extend known information on bimolecular triplexes by examining the effect of a variable CNC base triad (where N = A, C, T, or G) on a 10 base triad triplex that mimics the triplex motif in H-form DNA. We also examine the effect that a duplex extension of four base pairs has on triplex stability and selectivity for the base N. Results from thermal denaturation experiments indicate that the fully complementary triplex is more stable than its duplex counterpart (DeltaTm = 13 degrees C) and is resistant to degradation by bovine spleen phosphodiesterase for at least 24 h at 10 degrees C. A single-base mismatch in the purine strand of the triplex structure is destabilizing (DeltaTm = approximately 20 degrees C), and all structures containing a mismatch were readily degraded by bovine spleen phosphodiesterase. An extension of four duplex base pairs onto the triplex structure affects the stability of the DNA complex and may have implications relevant to H-form DNA.