No abstract

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.

Vesicle polydispersity and leakage of solutes from the vesicle lumen influence the measurement and analysis of osmotically induced vesicle swelling and lysis, but their effects have not been considered in previous studies of these processes. In this study, a model is developed which expressly includes polydispersity and leakage effects. The companion paper demonstrated the preparation and characterization of large unilamellar lipid vesicles. A dye release technique was employed to indicate the leakage of solutes from the vesicles during osmotic swelling. Changes in vesicle size were monitored by dynamic light scattering (DLS). In explaining the results, the model identifies three stages. The first phase involves differential increases in membrane tension with strain increasing in larger vesicles before smaller ones. In the second phase, the yield point for lysis (leakage) is reached sequentially from large sizes to small sizes. In the final phase, the lumen contents and the external medium partially equilibrate under conditions of constant membrane tension. When fit to the data, the model yields information on polydispersity-corrected values for membrane area compressibility, Young's modulus, and yield point for lysis.

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit 0 ), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit 6 ), human component C1 (hMBP-Cit 0 ), and human component C8 (hMBP-Cit 6 ), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit 0 and hMBP-Cit 0 bound CaM in a Ca 2+-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K d ‫ס‬ 144 ± 76 nM for rmMBP-Cit 0 , and K d ‫ס‬ 42 ± 15 nM for hMBP-Cit 0 . For rmMBP-qCit 6 and hMBP-Cit 6 , the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination. Keywords:Myelin basic protein; calmodulin; multiple sclerosis; deimination; citrulline; intrinsic Trp fluorescence; fluorescence lifetime; dynamic light scattering; circular dichroism Myelin basic protein (MBP) is one of the most abundant proteins of the myelin sheath of the central nervous system (CNS), and its primary role is generally considered to be maintenance of the stability of the sheath by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane (Smith 1992;Moscarello 1997). However, the MBP family comprises numerous developmentally regulated isoforms, of which the 18.5-kD species is the most abundant in adult human myelin, and has been the most studied (Givogri et al. 2000(Givogri et al. , 2001. This isoform itself undergoes a complex series of posttranslational modifications, giving rise to charge isomers designated as components C1 to C8 (Moscarello 1997;Wood and Moscarello 1997;Zand et al. 1998). In addition to its associations with lipids, MBP Reprint requests to: George Harauz, Department of Molecular Biology and Genetics, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada; e-mail: gharauz@uoguelph.ca; fax: (519) 837-2075. 4 These authors contributed equally to this work. Article and publication are at http://www.proteinscience.org/cgi