The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.

It has been postulated that the control of thrombus formation on molecularly engineered surfaces is an important step in developing clinically durable small-diameter vascular prostheses. This has led to designing a membrane-mimetic assembly that contains physiological regulators of blood coagulation, thrombomodulin (TM) and heparin, to provide strategies for generating actively antithrombogenic surfaces. The membrane-mimetic construct contains polymeric phospholipid monolayer on an alkylated polyelectrolyte multilayer supported by planar substrate such as glass or silicone. When incorporated with TM, the model platform exhibited the biological function by catalyzing activation of protein C. Surface TM activity was extensively investigated at physiologic shear rates (50 sec-1 and 500 sec-1). Significantly, reaction rates become saturated at TM surface densities greater than or equal to ~ 800 fmole/cm2 due to due to a transport limitation. Based on the similar membrane-mimetic construct, a functional heparinized surface was designed as an alternative anticoagulant system. Immobilization of heparin onto membrane-mimetic surfaces was achieved through biotin-streptavidin binding specificity. Activity of surface heparin to facilitate thrombin inactivation was investigated at shear rates of 50 and 500 sec-1. Significantly, rate of thrombin decay becomes saturated when the surface coverage of heparin is higher than 4.4 pmole of heparin per cm2. We further investigated the effects of surface bound TM and heparin on tissue factor (TF) -induced thrombin generation in a flow model. Specifically, TF positioned over a 2 x 6 mm2 upstream region as a trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream (34 x 6 mm2) portion of the test film. Compared to TF alone surface, thrombin generation was profoundly reduced in the presence of surface bound TM and/or heparin. Significantly, thrombin production was maximally inhibited more than 85% in the presence of TM and heparin, possibly due to anticoagulant synergism of both anticoagulants. We believe that current membrane-mimetic systems can potentially create actively antithrombogenic surfaces.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid–protein interactions, membrane protein function and membrane structure in vitro. This dissertation describes a route to leverage the programmability of DNA nanotechnology to create DNA-encircled bilayers (DEBs), a novel nano-scaled membrane-mimetic system. DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. This straight-forward and robust route enables the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins. Next, we optimized the DEB system to provide proper anchoring of a large variety of lipids by creating an improved DNA scaffold. This scaffold, called DNA double-decker, consists of two interconnected DNA minicircles stacked on top of each other. In comparison to the DNA minicircle in DEB system, this scaffold is two times thicker and contains two times more hydrophobic strands, which should increase the stability of the lipid bilayer rim. Finally, we explored the option of using DEBs in studies of GPCRs using CCR5 as a model protein. The CCR5 was labeled with DNA strands, purified and characterized. The strands on CCR5 are complementary to the protruding strands on the DNA minicircle in DEBs. This can allow the reconstitution of GPCRs inside DEBs with controlled orientation of the receptor.