Control of protein activity by photoinduced spin polarized charge reorganization.

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

CCR7 signalosomes are preassembled on tips of lymphocyte microvilli in proximity to LFA-1.

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

Long-Range Charge Reorganization as an Allosteric Control Signal in Proteins.

A new mechanism of allostery in proteins, based on charge rather than structure, is reported. We demonstrate that dynamic redistribution of charge within a protein can control its function and affect its interaction with a binding partner. In particular, the association of an antibody with its target protein antigen is studied. Dynamic charge shifting within the antibody during its interaction with the antigen is enabled by its binding to a metallic surface that serves as a source for electrons. The kinetics of antibody-antigen association are enhanced when charge redistribution is allowed, even though charge injection happens at a position far from the antigen binding site. This observation points to charge-reorganization allostery, which should be operative in addition or parallel to other mechanisms of allostery, and may explain some current observations on protein interactions.

Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time (<τs >) of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

Single-molecule Spectroscopy: Exploring Heterogeneity in Chemical and Biological Systems.

Many chemical and biological systems are heterogeneous in the molecular length scale (∼ 1 nm). Heterogeneity in many chemical systems and organized assemblies may be monitored using single-molecule spectroscopy (SMS). In SMS, the size of the focal spot (i.e., the smallest region to be probed) is nearly half of the excitation wavelength (λ/2, i.e., 200-375 nm) for visible light (400-750 nm). We discuss how one can get spatial resolutions better than 200 nm using molecules as nanometric probes. We show that polymer hydrogels, lipid vesicles, room temperature ionic liquids (RTILs), and binary liquid mixtures exhibit such heterogeneity. Another important observation is solute-dependent friction in RTILs. In an RTIL, diffusion of an ionic solute is slower than that of a neutral solute.

Unfolding and refolding of a protein by cholesterol and cyclodextrin: a single molecule study.

Unfolding/refolding of a plasma protein, human serum albumin (HSA), is studied using fluorescence correlation spectroscopy (FCS) and single molecule fluorescence resonance energy transfer (sm-FRET). Addition of cholesterol causes unfolding of HSA resulting in an increase in the hydrodynamic diameter (dH = 2rH) from 76 Å in the native state to 120 Å upon addition of 1 mM cholesterol. Addition of ß-cyclodextrin to HSA (unfolded by cholesterol) restores the hydrodynamic diameter back to 78 Å. The cholesterol induced unfolding and ß-cyclodextrin induced refolding are also monitored by measuring the distance between a FRET donor (CPM dye, D) and a FRET acceptor (Alexa 488, A) covalently attached to the protein (HSA). It is observed that the average D-A distance increases from 45 ± 1 Å at 0 mM cholesterol to 51 ± 1 Å at 1 mM cholesterol. Upon addition of ß-cyclodextrin, the D-A distance is restored to 45 ± 1 Å. The binding study indicates that nearly 94% of HSA molecules remain bound to cholesterol in the absence of ß-cyclodextrin and only 5% binds to cholesterol in the presence of ß-cyclodextrin. As much as 57% of the HSA and 99% of the cholesterol molecules bind to ß-cyclodextrin. Thus ß-cyclodextrin removes cholesterol from HSA by hydrophobic binding to cholesterol ("strip off") and also, itself binds to HSA. The conformational dynamics results suggest that addition of ß-cyclodextrin restores native like binding free energy and folding dynamics.

Ionic liquid induced dehydration and domain closure in lysozyme: FCS and MD simulation.

Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the structure and dynamics of the protein, lysozyme, is investigated by fluorescence correlation spectroscopy (FCS) and molecular dynamic (MD) simulation. The FCS data indicate that addition of the RTIL ([pmim][Br]) leads to reduction in size and faster conformational dynamics of the protein. The hydrodynamic radius (rH) of lysozyme decreases from 18 Å in 0 M [pmim][Br] to 11 Å in 1.5 M [pmim][Br] while the conformational relaxation time decreases from 65 µs to 5 µs. Molecular origin of the collapse (size reduction) of lysozyme in aqueous RTIL is analyzed by MD simulation. The radial distribution function of water, RTIL cation, and RTIL anion from protein clearly indicates that addition of RTIL causes replacement of interfacial water by RTIL cation ([pmim](+)) from the first solvation layer of the protein providing a comparatively dehydrated environment. This preferential solvation of the protein by the RTIL cation extends up to ∼30 Å from the protein surface giving rise to a nanoscopic cage of overall radius 42 Å. In the nanoscopic cage of the RTIL (42 Å), volume fraction of the protein (radius 12 Å) is only about 2%. RTIL anion does not show any preferential solvation near protein surface. Comparison of effective radius obtained from simulation and from FCS data suggests that the "dry" protein (radius 12 Å) alone diffuses in a nanoscopic cage of RTIL (radius 42 Å). MD simulation further reveals a decrease in distance ("domain closure") between the two domains (alpha and beta) of the protein leading to a more compact structure compared to that in the native state.

Effect of NaCl on ESPT-mediated FRET in a CTAC micelle: a femtosecond and FCS study.

Femtosecond upconversion, single-molecule fluorescence resonance energy transfer (sm-FRET) and fluorescence correlation spectroscopy (FCS) are applied to study the competition between excited-state proton transfer (ESPT) and FRET [to rhodamine 6G (R6G)] of 8-hydroxypyranine-1,3,6-trisulfonate (HPTS) in cetyltrimethylammonium chloride (CTAC) micelles. Pyranine exhibits dual emission at λ(em)=430 nm for ROH and 520 nm for RO(-). The absorption spectrum of R6G (acceptor) has very good overlap with the RO(-) emission and poor overlap with ROH emission. It is observed that FRET occurs readily from the RO(-)* state of HPTS (donor) to R6G (acceptor). Multiple timescales of FRET were detected from the rise time of acceptor emission. The different timescales correspond to different donor-acceptor distances. The ultrafast components (8.5 and 13 ps) are assigned to FRET at a close contact of donor and acceptor (≈20 Å). The longer components (500 and 800 ps) arise from long-distance FRET from the donor to the acceptor (≈40 Å) located in different regions of the CTAC micelle. The larger donor-acceptor distances agree with those obtained from an sm-FRET study. On addition of 4 M NaCl to CTAC, the rate of proton transfer (k(PT)) slowed by about eight and two times, respectively, for the fast and slow sites of the CTAC micelle. As a result, the intensity of the ROH emission increases and that of RO(-) decreases. The decrease in the intensity of the RO(-) emission causes a decrease in the efficiency of FRET.

Dynamics in cytoplasm, nucleus, and lipid droplet of a live CHO cell: time-resolved confocal microscopy.

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ~34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time (<τs>) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (<τs> = 750 ± 50 ps) and cytoplasm (<τs> = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ~ 11.2 cP, ε ~ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ~ 21.7 cP, ε ~ 7.11).

Solvation dynamics of biological water in a single live cell under a confocal microscope.

Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell. A DNA binding dye, DAPI, is found to be inside the nucleus of the cell. The locations of the probes are clearly seen in the image. Emission maxima of the dyes (DCM in cytoplasm and DAPI in the nucleus) are compared to those of the same dyes in different solvents. From this, it is concluded that the polarity (dielectric constant, ε) of the microenvironment of DCM in the cytoplasm is ~15. The nucleus is found to be much more polar with ε ≈ 60 (as reported by DAPI). The diffusion coefficient (and hence viscosity) in the cytoplasm and the nucleus was determined using fluorescence correlation spectroscopy (FCS). The diffusion coefficient (D(t)) of the dye (DCM) in the cytoplasm is 2 µm(2) s(-1) and is ~150 times slower than that in bulk water (buffer). D(t) of DAPI in the nucleus (15 µm(2) s(-1)) is 30 times slower than that in bulk water (buffer). The extremely slow diffusion inside the cell has been ascribed to higher viscosity and also to the binding of the probes (DCM and DAPI) to large biological macromolecules. The solvation dynamics of water in the cytoplasm (monitored by DCM) exhibits an average relaxation time [τ(sol)] of 1250 ± 50 ps, which is about 1000 times slower than in bulk water (1 ps). The solvation dynamics inside the nucleus (studied using DAPI) is about 2-fold faster, [τ(sol)] ≈ 775 ps. The higher polarity, faster diffusion, and faster solvation dynamics in the nucleus indicates that it is less crowded and less restricted than the cytoplasm.

Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: fluorescence correlation spectroscopy.

Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Proteins with Respect to Cellular Surface Topography.

We describe microvillar cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, the need for carbon coating, and immunogold clustering, and is amenable to future developments involving, for example, live-cell imaging. This protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell-surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

Unraveling the Interaction of Diflunisal with Cyclodextrin and Lysozyme by Fluorescence Spectroscopy.

Understanding the interaction between the drug:carrier complex and protein is essential for the development of a new drug-delivery system. However, the majority of reports are based on an understanding of interactions between the drug and protein. Here, we present our findings on the interaction of the anti-inflammatory drug diflunisal with the drug carrier cyclodextrin (CD) and the protein lysozyme, utilizing steady-state and time-resolved fluorescence spectroscopy. Our findings reveal a different pattern of molecular interaction between the inclusion complex of ß-CD (ß-CD) or hydroxypropyl-ß-CD (HP-ß-CD) (as the host) and diflunisal (as the guest) in the presence of protein lysozyme. The quantum yield for the 1:2 guest:host complex is twice that of the 1:1 guest:host complex, indicating a more stable hydrophobic microenvironment created in the 1:2 complex. Consequently, the nonradiative decay pathway is significantly reduced. The interaction is characterized by ultrafast solvation dynamics and time-resolved fluorescence resonance energy transfer. The solvation dynamics of the lysozyme becomes 10% faster under the condition of binding with the drug, indicating a negligible change in the polar environment after binding. In addition, the fluorescence lifetime of diflunisal (acceptor) is increased by 50% in the presence of the lysozyme (donor), which indicates that the drug molecule is bound to the binding pocket on the surface of the protein, and the average distance between active tryptophan in the hydrophobic region and diflunisal is calculated to be approximately 50 Å. Excitation and emission matrix spectroscopy reveals that the tryptophan emission increases 3-5 times in the presence of both diflunisal and CD. This indicates that the tryptophan of lysozyme may be present in a more hydrophobic environment in the presence of both diflunisal and CD. Our observations on the interaction of diflunisal with ß-CD and lysozyme are well supported by molecular dynamics simulation. Results from this study may have an impact on the development of a better drug-delivery system in the future. It also reveals a fundamental molecular mechanism of interaction of the drug-carrier complex with the protein.

Solvation dynamics under a microscope: single giant lipid vesicle.

Picosecond spectroscopy under a confocal microscope is employed to study solvation dynamics of coumarin 153 (C153) inside a single giant lipid vesicle (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC) of diameter 20 µm. Fluorescence correlation spectroscopy (FCS) indicates that the diffusion coefficient (D(t)) of the probe (coumarin153, C153) in the immobilized vesicle displays a wide distribution from ~3 to 21 µm(2) s(-1). The distribution of D(t) suggests that the microenvironment of the probe (C153) is highly heterogeneous and the local friction is different for probe molecules in different regions. The values of D(t) is significantly smaller than that for the same dye in bulk water (550 µm(2) s(-1)). This suggests that the probe is located in the interface or membrane region rather than in the water pool of the vesicle. The solvation time of C153 in different regions of the lipid vesicle varies between 750 to 1200 ps. This result clearly shows that a confocal microscope is able to resolve the spatial heterogeneity in local friction (i.e., D(t)) and solvation dynamics within a lipid vesicle.

Diffusion of organic dyes in a niosome immobilized on a glass surface using fluorescence correlation spectroscopy.

Giant multilameller niosomes containing cholesterol and triton X-100 are studied using fluorescence correlation spectroscopy (FCS). Dynamic light scattering (DLS) data indicates formation of niosomes of broadly two different sizes (diameter)--~150 nm and ~1300 nm. This is confirmed by field emission scanning electron microscopy (FE-SEM) and confocal microscopy. The diffusion coefficient (D(t)) of three organic dyes in the niosome immobilized on a glass surface is studied using fluorescence correlation spectroscopy. On addition of the room temperature ionic liquids (RTIL) (1-methyl-3-pentylimidazolium bromide, [pmim][Br] and 1-methyl- 3-pentylimidazolium tetra-fluoroborate, [pmim][BF(4)]) the size of the niosome particles increases. The D(t) of all the organic dyes (DCM, C343 and C480) increases on addition of RTILs, indicating faster diffusion. The viscosity calculated from the D(t) of the three dyes exhibits weak probe dependence. Unlike lipid or catanionic vesicle, the D(t) values in a niosome exhibit very narrow distribution. This indicates that the niosomes are fairly homogeneous with small variation of viscosity.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation.

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαß and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαß as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.

Preferential targeting of i-motifs and G-quadruplexes by small molecules.

i-Motifs and G-quadruplexes are dynamic nucleic acid secondary structures, which are believed to play key roles in gene expression. We herein report two peptidomimetic ligands (PBP1 and PBP2) that selectively target i-motifs and G-quadruplexes over double-stranded DNA. These peptidomimetics, regioisomeric with respect to the position of triazole/prolinamide motifs, have been synthesized using a modular method involving Cu(i)-catalyzed azide and alkyne cycloaddition. The para-isomer, PBP1 exhibits high selectivity for i-motifs while the meta-isomer PBP2 binds selectively to G-quadruplex structures. Interestingly, these ligands have the ability to induce G-quadruplex or i-motif structures from the unstructured single-stranded DNA conformations, as observed using single molecule Förster resonance energy transfer (smFRET) studies. The quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase assays indicate that PBP1 upregulates and PBP2 downregulates BCL-2 gene expression in cancer cells.

Small molecule regulated dynamic structural changes of human G-quadruplexes.

The changes in structure and dynamics of oncogenic (c-MYC) and telomeric (h-TELO) G-rich DNA sequences due to the binding of a novel carbazole derivative (BTC) are elucidated using single-molecule Förster resonance energy transfer (sm-FRET), fluorescence correlation spectroscopy (FCS) and NMR spectroscopy. In contrast to the previous reports on the binding of ligands to pre-folded G-quadruplexes, this work illustrates how ligand binding changes the conformational equilibria of both unstructured G-rich DNA sequences and K+-folded G-quadruplexes. The results demonstrate that K+ free c-MYC and h-TELO exist as unfolded and partially folded conformations. The binding of BTC shifts the equilibria of both investigated DNA sequences towards the folded G-quadruplex structure, increases the diffusion coefficients and induces faster end-to-end contact formation. BTC recognizes a minor conformation of the c-MYC quadruplex and the two-tetrad basket conformations of the h-TELO quadruplex.

Solvation dynamics and intermittent oscillation of cell membrane: live Chinese hamster ovary cell.

Dynamics of the exofacial thiols (i.e., cell surface thiol containing membrane proteins) of a live Chinese hamster ovary (CHO) cell is probed by time-resolved confocal microscopy. For this purpose, a fluorescent probe, 7-(diethylamino)-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) is covalently attached to the exofacial thiols. The emission maximum of CPM bound exofacial thiols indicates a highly exposed and polar environment. Using CPM, we studied solvation dynamics, for the first time, at the membrane of a live cell. The thiol containing membrane proteins shows ultraslow response with average solvation time, ⟨τs⟩ = 475 ps. CPM labeled exofacial thiols also show spontaneous, intermittent oscillation in fluorescence intensity with a period of 0.5-1.0 s. This is ascribed to reversible, intermittent changes in the structure and conformation of the membrane proteins.