Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with pico-Newton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ~2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.
In vitro analysis confirms talin binding is sufficient for activation and extension of membrane-embedded integrin.
The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are watersoluble, monodisperse self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 Å and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 Å, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.
G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated lightactivated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.Visual phototransduction is quenched by a two-step mechanism. First, light-activated rhodopsin (Rh*) 3 is phosphorylated multiple times by GRK1. Arrestin-1 4 binding to active phosphorylated rhodopsin (P-Rh*) blocks further transducin activation (1) by steric exclusion (2). The binding of arrestin-1 to P-Rh* is an important molecular mechanism for signal shut-off (3). However, key details of the requirements for physical interaction of arrestin-1 with rhodopsin remain to be explored. Rhodopsin, which is highly concentrated in photoreceptor membranes, has been observed to form arrays of dimers, thus raising the possibility that the dimer is a functional unit (4). Although evidence of a preferred dimer interface has been reported (5), the functional role of rhodopsin oligomers remains controversial (6, 7). Accumulating evidence with other GPCRs indicates that oligomerization could be widespread (8 -10). Several models for rhodopsin dimers (11, 12) and monomers (6, 13) interacting with signaling partners transducin, rhodopsin kinase, and arrestin have been proposed. These models need rigorous experimental testing at different steps of the functional cycle of rhodopsin and other class A GPCRs (reviewed in Refs. 7 and 14). Modified high density lipoprotein particles (nanodiscs) consist of a phospholipid bilayer stabilized by a membrane scaffold protein (MSP) (15-17). These nanodiscs can be used to selectively isolate monomeric GPCRs imbedded into lipid bilayer. We (18) and others (19,20) have previously demonstrated that rhodopsin monomers incorporated in nanodiscs are highly functional in signaling to transducin. The same was shown for monomeric rhodopsin purified in detergent (dodecyl maltoside), where rhodopsin and transducin form a 1:1 complex (21). Howe...
Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan £uorescence and di¡erential scanning calorimetry. Laurdan is a £uorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity pro¢le can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Di¡erential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3^4 ‡C higher for both lipids when incorporated into Nanodiscs. These ¢ndings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins. ß
Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450. Cytochrome P450 3A4 (CYP3A4)2 is the most prevalent cytochrome P450 in human liver and is responsible for the breakdown of numerous xenobiotics, including more than 50% of the currently marketed pharmaceuticals (1, 2). Most cytochromes P450 operate via a catalytic cycle wherein atmospheric dioxygen is ligated to the ferrous heme of the P450 protein and is reduced through electron input from pyridine nucleotide oxidation as provided by an associated electron transfer chain. In the case of CYP3A4 this is an 80-kDa reductase molecule containing both FAD and FMN prosthetic groups. A central intermediate in this reaction cycle is the ferrous-dioxygen adduct generated through ferric-ferrous reduction of the heme center and dioxygen binding. This intermediate, the "oxy complex" can either decay via release of superoxide and regeneration of the ferric heme (3) or be reduced with a second redox equivalent resulting in the cleavage of the oxygen-oxygen bond and generation of a higher valence metal-oxo complex that can initiate radical chemistry and subsequent substrate oxygenation. The former autoxidation pathway represents the first branching point between productive and unproductive pathways in P450 catalysis wherein reducing equivalents leak into the production of unwanted reduced oxygen species (4 -7). Subsequent uncoupling channels include the release of the two electron-reduced dioxygen as peroxide and a further reduction of the higher valence metal-oxo complex to produce a second water molecule.The oxy-ferrous intermediate in various cytochromes P450 was documented many decades ago (3, 8 -11). For the soluble bacterial systems, the autoxidation process was shown to be first order (3,8,9) but for the membrane-associated isozymes was often d...
KRas4b is a small G-protein whose constitutively active oncogenic mutants are present in 90% of pancreatic cancers. Using fully post-translationally modified KRAS4b, we investigated the role of lipid identity in the recruitment of KRas4b to a membrane surface of defined composition. Application of a newly developed single frequency fluorescence anisotropy decay experiment to this system revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP2. We conducted molecular dynamics simulations to look for an origin of this specificity. In the case of membranes containing PIP2 the protein formed long-lived salt bridges with PIP2 head groups but not the monovalent DMPS, explaining the experimentally observed lipid specificity. Additionally, we report that PIP2 forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient the protein in a manner expected to facilitate association with upstream and downstream signaling partners.
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