@article{chekmenev_gor’kov_cross_alaouie_smirnov_2006, title={Flow-Through Lipid Nanotube Arrays for Structure-Function Studies of Membrane Proteins by Solid-State NMR Spectroscopy}, volume={91}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33749506571&partnerID=MN8TOARS}, DOI={10.1529/biophysj.106.085191}, abstractNote={A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60-mum-long nanopores were employed to form macroscopically aligned peptide-containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a period of up to a few hours and temperatures as high as 70 degrees C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid-state 19.6 T (17)O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu(10) carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the (17)O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMR-based studies, including structure-function, drug screening, proton exchange, pH, and other titration experiments, will benefit significantly from the method described here.}, number={8}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Chekmenev, Eduard Y. and Gor’kov, Peter L. and Cross, Timothy A. and Alaouie, Ali M. and Smirnov, Alex I.}, year={2006}, month={Oct}, pages={3076–3084} }
 @article{alaouie_smirnov_2006, title={Formation of a ripple phase in nanotubular dimyristoylphosphatidylcholine Bilayers confined inside nanoporous aluminum oxide substrates observed by DSC}, volume={22}, ISSN={["0743-7463"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33745760734&partnerID=MN8TOARS}, DOI={10.1021/la060448z}, abstractNote={Phase properties of substrate-supported nanotubular dimyristoylphosphatidylcholine (DMPC) bilayers confined within nanoporous channels of anodic aluminum oxide were characterized by DSC and compared with unsupported vesicles. In addition to the main phase transition, all samples exhibited a pretransition with a characteristic midpoint hysteresis between heating and cooling scans. The pretransition indicates that nanotubular bilayers could exist in a ripple phase, whereas hysteresis points to a similarity in the phase transition mechanisms. Observance of the ripple phase in lipid nanotubes is an indication of fully hydrated and only slightly perturbed bilayer surface.}, number={13}, journal={LANGMUIR}, publisher={American Chemical Society (ACS)}, author={Alaouie, Ali M. and Smirnov, Alex I.}, year={2006}, month={Jun}, pages={5563–5565} }
 @article{alaouie_smirnov_2006, title={Ultra-stable temperature control in EPR experiments: Thermodynamics of gel-to-liquid phase transition in spin-labeled phospholipid bilayers and bilayer perturbations by spin labels}, volume={182}, ISSN={["1090-7807"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33749070613&partnerID=MN8TOARS}, DOI={10.1016/j.jmr.2006.07.002}, abstractNote={An ultra-stable variable temperature accessory for EPR experiments with biological samples has been designed and tested. The accessory is comprised from a digitally controlled circulator bath that pumps fluid through high-efficiency aluminum radiators attached to an EPR resonator of a commercial X-band EPR spectrometer. Temperature stability of this new accessory after a 15 min re-equilibration is at least ±0.007 K. For a standard 1-cm-long capillary sample arranged inside an EPR tube filled with silicon oil, the temperature variations do not exceed ±0.033 K over the sample temperature range from 283 to 333 K. This new accessory has been tested by carrying out a comparative spin-labeling EPR and differential scanning calorimetry (DSC) study of the gel-to-liquid phase transition in multilamellar vesicles (MLV) composed of a synthetic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We demonstrate that the gel-to-liquid phase transition temperatures of MLV DMPC measured by EPR and DSC agree within ±0.02 K experimental error even though the sample for EPR study was labeled with 1 mol% of 5PC (1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3 phosphocholine). Cooperative unit number measured by EPR, N = 676 ± 36, was almost 50% higher than that obtained from DSC (N = 458 ± 18). These high values of N indicate that (i) the lipid domains should include at least several spin-labeled lipid molecules and (ii) the spin-probe 5PC molecules are not excluded into domains that are different from the bulk lipid phase as was speculated earlier. Overall, our data provide DSC and EPR evidence that in studies of the gel-to-liquid phase transition, the effect of bilayer perturbation by spin-labeled lipids is negligible and therefore thermodynamic parameters of the phase transition can be accurately measured by spin-labeling EPR. This might serve as an indication when spin-labeled molecules with structures similar to those of lipids are introduced at low concentrations, they are easily accommodated by fluid phospholipid bilayers without significant losses of the lipid cooperativity.}, number={2}, journal={JOURNAL OF MAGNETIC RESONANCE}, publisher={Elsevier BV}, author={Alaouie, Ali M. and Smirnov, Alex I.}, year={2006}, month={Oct}, pages={229–238} }
 @article{alaouie_smirnov_2005, title={Cooperativity and kinetics of phase transitions in nanopore-confined bilayers studied by differential scanning calorimetry}, volume={88}, ISSN={["1542-0086"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-21244463058&partnerID=MN8TOARS}, DOI={10.1529/biophysj.104.056523}, abstractNote={The first-order nature of the gel-to-liquid crystal phase transition of phospholipid bilayers requires very slow temperature rates in differential scanning calorimetry (DSC) experiments to minimize any rate-dependent distortions. Proportionality of the DSC signal to the rate poses a problem for studies of substrate-supported bilayers that contain very small volumes of the lipid phase. Recently, we described lipid bilayers self-assembled inside nanoporous substrates. The high density of the nanochannels in these structures provides at least a 500-fold increase in the bilayer surface area for the same size of the planar substrate chips. The increased surface area enables the DSC studies. The rate-dependent DSC curves were modeled as a convolution of a conventional van't Hoff shape and a first-order decay curve of the lipid rearrangement. This analysis shows that although confinement of bilayers to the nanopores of approximately 177 nm in diameter results in a more than threefold longer characteristic time of the lipid rearrangement and a decrease in the cooperative unit number, the phase transition temperature is unaffected.}, number={2}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Alaouie, AM and Smirnov, AI}, year={2005}, month={Feb}, pages={L11–L13} }