PMID: 39621;Abstract:
The low field portion of the 360 MHz 1H nuclear magnetic resonance spectrum of phenylmethanesulfonyl-subtilisin Novo (EC 3.4.21.14) has been studied as a function of pH. Analysis of the pH-induced chemical shift changes occurring between 6 to 7 ppm revealed five classes of ionizable residues with pK values (uncorrected) of 10.3, 10.7, 10.7, 10.8, and 11.0. With a single exception, the titration curves can be fit by assuming a simple proton ionization equilibrium. Four classes of low intensity broad resonances, assigned to the histidyl residues, are observed between 8 and 9 ppm. Uncorrected pK values of 5.4, 5.7, 6.0, and 6.4 were determined for the residues comprising each of these classes. The spectral data are consistent with protonation of one or more histidyl residues upon acid induced denaturation of the protein. These results are compared with those of analogues studies performed by the use of other techniques. © 1979.
PMID: 21190661;PMCID: PMC3010004;Abstract:
Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (SCD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins. © 2011 by the Biophysical Society.
