Pentameric Units Research Articles

A broadly reactive ultralong bovine antibody that can determine the integrity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)'s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.

Uranyl-Tc(VII)/Tc(V) hybrid clusters.

Pertechnetate (99TcVIIO4-), reduced Tc, and actinides co-exist in spent nuclear fuel and legacy wastes. They co-transport in fuel reprocessing and waste disposal scenarios, necessitating an understanding of co-speciation. Here, we report five new molecular cluster/framework structures with pentameric and tetrameric uranyl building units decorated by TcO4-/ReO4- oxoanions, or fused with the reduced technetyl cation Tc(V)O. The latter, obtained by Tc auto-reduction (without the intentional introduction of a reducing environment), broadens the basis for Tc-reduction and the burgeoning polyoxometalate-like behavior of technetium.

In silico investigation of symptom development model based on coat protein interactions of two cucumber mosaic virus strains

Cucumber mosaic virus (CMV) causes serious diseases in agricultural crops worldwide, predominantly in vegetable and ornamental plants. A critical interaction has been reported between the plant Ferredoxin I (Fd I) protein with the coat protein (CP) of the M strain of CMV (M-CMV) but not by the CP of the Q strain of CMV (Q-CMV). To ascertain the most likely symptom development model based on CMV CP interacting with the plant Fd I protein, further in silico investigation was performed on the CPs of the strains Q-CMV and M-CMV. The biological assembly of the CMV CP in virions is composed of hexameric and pentameric units. The results showed that there were significantly different interactions between pentameric and hexameric forms of M and Q-CMV CPs with Fd I. Pentameric structural forms of M-CMV CP interact strongly with Fd I compared to pentameric structures of Q-CMV CP, while there were no significant differences in the affinities of hexameric structures for Fd I between M and Q-CMV CPs. it can be deduced that probably pentameric structures of M-CMV CP block activation of plant Fd I protein. Therefore, it could be hypothesized that trapping Fd I in the complex of pentameric structures can lead to inhibition of the activity of plant Fd I in the photosynthetic electron transport chain leading the severe symptom development seen in M-CMV infected plants but not in Q-CMV infected plants. It seems likely that any factor that inhibits the interaction of CMV CPs with plant Fd I can prevent this symptom development.

The Polarizable continuum potential model in the liquid state Calculations. Density functional computational analysis of the anharmonic vibrational spectrum of liquid water

A Density Functional calculation, using the Gaussian 03 W protocol with the B3LYP functional and the SCRF-Polarizable Continuum model (SCRF-PCM), has been performed on a pentameric unit, constituted by a central water molecule tetrahedrically coordinated to four other surrounding molecules by hydrogen bond (HB) interactions. The calculation has been aimed at reproducing vibrational distributions of the intra-molecular and intermolecular modes, which contribute at widening the stretching and bending OH bands in the liquid vibrational spectrum, and those at low frequencies, commonly considered intermolecular motions of O-H…O stretching and bending type, in the HB tetrahedral network of liquid water. The so called librational large distribution has been also nicely reproduced by this calculation. Energetic and geometrical considerations have been discussed for the equilibrium structure of this pentameric assembly, representing the basic unit presently used to simulate more space-extended structures of liquid water. These are constituted by different domains of ordered (open, or ice-like) and unordered (closed) patches of water clusters. Although the small cluster used in this calculation represents only an approximation to treat the real organization existing in the liquid water complex system, our results seems to reproduce quite well both the vibrational spectrum and the energetic and geometric situations for this very important liquid.

Configurations of V4+ centers in the MoVO catalyst material. A systematic stability analysis of DFT results

The reactivity of a catalyst is in part determined by its geometric and electronic structure. Here we present a model that is able to describe the energy trend of the important oxidation catalyst material MoVO, as obtained from hybrid density functional calculations for various V4+/V5+ configurations. For an exemplary V/Mo occupancy, we systematically examined the universe of all V4+ distributions. The distribution of these V4+ centers, in combination with the induced lattice distortions, plays a key role in determining the stability of the material, entailing energy variations of up to ~140 kJ mol−1 per unit cell. Hence, for this kind of catalyst, it is crucial to account for the V4+ distributions. To this end, we are proposing novel predictive models based on features like the number of Mo centers with two reduced neighbors V4+ and the locations of potentially reducible centers V5+. For the V/Mo occupancy chosen, these models are able to describe the energy variation due to the V4+ distribution with root mean square errors as low as 6 kJ mol−1. Accordingly, catalytically selective sites featuring pentameric units with a single polaron center are among the most of stable configurations. Another aspect of this work is to understand energy contributions of polaron arrangements bracketing Mo centers.

HER3-targeted protein chimera forms endosomolytic capsomeres and self-assembles into stealth nucleocapsids for systemic tumor homing of RNA interference in vivo.

RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.

Acrolein oxidation to acrylic acid over the MoVOx material. Insights from DFT modeling

MoVOx-type catalysts are very active and very selective in the commercially important oxidation of acrolein to acrylic acid. To contribute to unravelling the thus far poorly understood mechanism, we carried out hybrid DFT calculations on embedded cluster models, focusing on the so-called pentameric unit as active site. In the absence of water, we calculated a large energy span, 169 kJ mol–1, for this transformation. After addressing the hydrolysis of the surface, we examined four scenarios of the acrolein conversion to explore the effect of water. The most favorable variant has a notably lower energy span, 146 kJ mol–1, in line with experimental observations that indicate a strong acceleration of the catalytic transformation in the presence of water.

Structure and properties of a novel staircase-like decanuclear [CuII10] cluster supported by carbonate and carboxylate bridges

A novel staircase-like decanuclear copper(ii) cluster composed of a pair of [CuII5] pentameric units that are linked together exclusively by two μ2:η2:η1carbonate ligands is reported. The cluster also shows a rare μ3:η2:η1:η1bridging coordination mode of benzoate groups of the ligand.

Structure and assembly of scalable porous protein cages

Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively. Remarkably, these expanded particles assume unprecedented tetrahedrally and icosahedrally symmetric structures constructed entirely from pentameric units. Large keyhole-shaped pores in the shell, not present in the wild-type capsid, enable diffusion-limited encapsulation of complementarily charged guests. The structures of these supercharged assemblies demonstrate how programmed electrostatic effects can be effectively harnessed to tailor the architecture and properties of protein cages.

Non-Enveloped Virus Entry: Structural Determinants and Mechanism of Functioning of a Viral Lytic Peptide

In the absence of lipid envelopes and associated fusion proteins, non-enveloped viruses employ membrane lytic peptides to breach the limiting membranes of host cells. Although several of these lytic peptides have been identified and characterized, their manner of deployment and interaction with host membranes remains unclear in most cases. We are using the gamma peptide of Flock House Virus (FHV), a model non-enveloped virus, to understand the mechanistic details of non-enveloped virus interaction with host cell membranes. We utilized a combination of biophysical assays, molecular dynamics simulation studies, and single-particle cryo-electron microscopy to elucidate the functional and structural determinants for membrane penetration by gamma in context of the FHV capsid. Although the amphipathic, helical N-terminal region of gamma (γ1) was previously thought to be the membrane-penetrating module, with the C-terminal region having a supporting role in correct structural positioning of γ1, we demonstrate that the C terminus of gamma directly participates in membrane penetration. Our studies suggest that full-length gamma, including the hydrophobic C terminus, forms an alpha-helical hairpin motif, and any disruption in this motif drastically reduces its functionality, in spite of the correct positioning of amphipathic γ1 in the virus capsid. Taken together, our data suggest that the most effective module for membrane disruption is a pentameric unit of full-length gamma, released from the virus, which associates with membranes via both N- and C-terminal ends.

Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

Further New Borosulfates: Synthesis, Crystal Structure, and Vibrational Spectra of A[B(SO4)2] (A = Na, K, NH4) and the Crystal Structures of Li5[B(SO4)4] and NH4[B(S2O7)2

AbstractThe reaction of alkali sulfates and boron oxide in oleum yielded the complexes A[B(S2O7)2] (A = Li, Na, K, NH4). This way we obtained the first ammonium‐containing borosulfate and we determined the crystal structure. NH4[B(S2O7)2] [Cc, Z = 8, a = 11.4403(2), b = 14.9439(2) c = 13.8693(2) Å] crystallizes isotyp to K[B(S2O7)2]. Thermal decomposition or treatment in vacuo resulted in compounds with composition A[B(SO4)2]. One‐dimensional chain structures are obtained for A = Na, K, NH4. NH4[B(SO4)2] [P4/ncc, Z = 4, a = 9.1980(1), c = 7.4258(1) Å] and K[B(SO4)2] [P4/ncc, Z = 4, a = 8.9739(3), c = 7.4114(3) Å] are isotyp with H3O[B(SO4)2]. The chains of edge‐sharing “BS4 super tetrahedra” have $\bar{4}$ symmetry. In Na[B(SO4)2] [P2/c, Z = 2, a = 5.434(5), b = 7.570(7) c = 7.766(8) Å, β = 108.56(3)°] the chain is twisted. Li5[B(SO4)4] [P21/c, Z = 4, a = 8.0191(1), b = 10.1211(1) c = 15.0401(2) Å, β = 104.033(1)°] contains isolated pentameric unit and represents a new structure type. Li5[B(SO4)4] was obtained from Li[B(S2O7)2] with additional Li2SO4/SO3. Vibrational spectra of A[B(S2O7)2] (A = Na, K, NH4) are presented and force constants calculated. In all structures BO4 and SO4 tetrahedra are connected by common apices forming BS4 super tetrahedra.

Synthesis and Structural Characterization of Hydrolysis Products within the Uranyl Iminodiacetate and Malate Systems

The interplay of hydrolysis and chelation by organic ligands results in the formation of novel uranium species in aqueous solutions. Many of these molecular complexes have been identified by spectroscopic and potentiometric techniques, but a detailed structural understanding of these species is lacking. Identification of possible uranyl hydrolysis products in the presence of organic functional groups has been achieved by the crystallization of molecular species into a solid-state compound, followed by structural and chemical characterization of the material. The structures of three novel molecular complexes containing either iminodiacetate (ida) (Na3[(UO2)3(OH)3(ida)3]·8H2O (1)) or malate (mal) (K(pip)2[(UO2)3O(mal)3]·6H2O (2a) (pip = C4N2H12), (2b) (pip)3[(UO2)3O(mal)3]·H2O, and (pip)6[(UO2)11(O)4(OH)4(mal)6(CO3)2]·23H2O (3)) ligands have been determined by single-crystal X-ray diffraction and have been chemically characterized by IR, Raman, and NMR spectroscopies. A major structural component in compounds 1 and 2 is a trimeric 3:3 uranyl ida or mal species, but different bridging groups between the metal centers create variations in the structural topologies of the molecular units. Compound 3 contains a large polynuclear cluster with 11 U atoms, which is composed of trimeric and pentameric building units chelated by mal ligands and linked through hydroxyl groups and carbonate anions. The characterized compounds represent novel structural topologies for U(6+) hydrolysis products that may be important molecular species in near-neutral aqueous systems.

Norovirus Capsid Proteins Self-Assemble through Biphasic Kinetics via Long-Lived Stave-like Intermediates

The self-assembly kinetics for a norovirus capsid protein were probed by time-resolved small-angle X-ray scattering and then analyzed by singular value decomposition and global fitting. Only three species contribute to the total scattering intensities: dimers, intermediates comprising some 11 dimers, and icosahedral T = 3 capsids made up of 90 dimers. Three-dimensional reconstructions of the intermediate robustly show a stave-like shape consistent with an arrangement of two pentameric units connected by an interstitial dimer. Upon triggering of self-assembly, the biphasic kinetics consist of a fast step in which dimers are assembled into intermediates, followed by a slow step in which intermediates interlock into capsids. This simple kinetic model reproduces experimental data with an excellent agreement over 6 decades in time and with nanometer resolution. The extracted form factors are robust against changes in experimental conditions. These findings challenge and complement currently accepted models for the assembly of norovirus capsids.

Exploring a New Structure Family: Alkali Borosulfates Na5[B(SO4)4], A3[B(SO4)3] (A = K, Rb), Li[B(SO4)2], and Li[B(S2O7)2

New alkali borosulfates were obtained by precipitation from oleum, solid-state reactions, or thermal decomposition. The crystal structures were characterized with single-crystal data. They are all based on corner-linked BO4 and SO4 tetrahedra with varying coordination of the alkali cations. According to the ratio of BO4 and SO4 tetrahedra, different frameworks are observed, i.e., noncondensed complex anions (1:4), one-dimensional chains (1:3), or three-dimensional (3D) networks (1:2). This is in analogy to silicates, where the ratio Si/O relates to the dimensionality also. For Na5[B(SO4)4], which exists in two different polymorphs, there are noncondensed pentameric units. Na5[B(SO4)4]-I: space group Pca21, a = 10.730(2) Å, b = 13.891(3) Å, c = 18.197(4) Å. Na5[B(SO4)4]-II: space group P212121, a = 8.624(2) Å, b = 9.275(2) Å, c = 16.671(3) Å. A3[B(SO4)3] (A = K, Rb) are isotypic with Ba3[B(PO4)3] adopting space group Ibca [K3[B(SO4)3], a = 7.074(4) Å, b = 14.266(9) Å, c = 22.578(14) Å; Rb3[B(SO4)3], a = 7.2759(5) Å, b = 14.7936(11) Å, c = 22.637(2) Å] with vierer chains of BO4tetrahedra based on two bridging and two terminal SO4 tetrahedra. Li[B(SO4)2] [space group Pc, a = 7.6353(15) Å, b = 9.342(2) Å, c = 8.432(2) Å, and β = 92.55(2)°] comprises a 3D network that is closely related to β-tridymite. Li[B(S2O7)2] [space group P212121, a = 10.862(2) Å, b = 10.877(2) Å, c = 17.769(4) Å] represents the first example of a disulfate complex with noncondensed [B(S2O7)2](-) units. Vibrational spectra were recorded from all compounds, and the thermal behavior was also investigated.

Xanthan chain length is modulated by increasing the availability of the polysaccharide copolymerase protein GumC and the outer membrane polysaccharide export protein GumB

Xanthan is a polysaccharide secreted by Xanthomonas campestris that contains pentameric repeat units. The biosynthesis of xanthan involves an operon composed of 12 genes (gumB to gumM). In this study, we analyzed the proteins encoded by gumB and gumC. Membrane fractionation showed that GumB was mainly associated with the outer membrane, whereas GumC was an inner membrane protein. By in silico analysis and specific globomycin inhibition, GumB was characterized as a lipoprotein. By reporter enzyme assays, GumC was shown to contain two transmembrane segments flanking a large periplasmic domain. We confirmed that gumB and gumC mutant strains uncoupled the synthesis of the lipid-linked repeat unit from the polymerization process. We studied the effects of gumB and gumC gene amplification on the production, composition and viscosity of xanthan. Overexpression of GumB, GumC or GumB and GumC simultaneously did not affect the total amount or the chemical composition of the polymer. GumB overexpression did not affect xanthan viscosity; however, a moderate increase in xanthan viscosity was achieved when GumC protein levels were increased 5-fold. Partial degradation of GumC was observed when only that protein was overexpressed; but co-expression of GumB and GumC diminished GumC degradation and resulted in higher xanthan viscosity than individual GumB or GumC overexpression. Compared with xanthan from the wild-type strain, longer polymer chains from the strain that simultaneously overexpressed GumB and GumC were observed by atomic force microscopy. Our results suggest that GumB-GumC protein levels modulate xanthan chain length, which results in altered polymer viscosity.

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.

Genetic, biological and structural hierarchies during sponge spicule formation: from soft sol–gels to solid 3D silica composite structures

Structural biomaterials are hierarchically organized and biofabricated. Although the structural complexity of most bioskeletons can be traced back from the millimeter-scale to the micrometer- or submicrometer-scale, the biological and/or genetic basis controlling the synthesis of these skeletons and their building blocks remained unknown. There is one distinguished example, the spicules of the siliceous sponges, for which the principle molecules and molecular-biological processes involved in their formation have been elucidated in the last few years. In this review, recent data on the different levels of molecular, biological and structural hierarchies controlling the synthesis of the picturesquely and intricately architectured spicules are summarized. The silicateins and their interacting/maturated proteins comprise the basic enzymatic/proteinous machinery that facilitates the polycondensation of silicate to biosilica. Two isoforms of silicatein, silicatein-α and silicatein-β, the enzyme that catalyzes the polymerization of orthosilicate to polymeric biosilica, have been identified. The remarkable feature of these enzymes is that, besides their enzymatic function, they act as structure-giving proteins that provide the platform for the organization of the silica spicules. Silicatein-α together with silicatein-β forms pentameric units that continue to grow in a linear pattern. The silicatein-interacting protein, silintaphin-1, stabilizes the initially formed silicatein fractals, while silintaphin-2 provides Ca2+ ions required for the appositional growth of the spicules. The biosilica formed during the enzymatically driven sol–gel process that is catalyzed by this multi-protein system is a soft, gel-like inorganic polymer. This soft biosilica undergoes a biologically controlled process of syneresis, resulting in a shrinkage of the silica network. During this reaction the biosilica is transformed into an elastic solid and gains the characteristic spicule morphology. A sizeable amount of protein, mostly silicatein, remains embedded in the biosilica material, thus forming a hybrid bioinorganic (“biosilica”) material. The process of syneresis involves the removal of water by cell-membrane-associated aquaporin channels and is guided by collagen bundles. Four cell types, sclerocytes, archaeocytes, chromocytes and lophocytes, participate in this structure-guiding process. In conclusion, this article attempts to overcome the frontiers in the understanding of the different levels of hierarchies, genetic, biological and structural, and to contribute towards the fabrication of new bioinspired functional materials.

Title:Stability of Viral Capsids and their Epitopes Using Multiscale Simulation

We investigate the stability of viral capsids and their individual pentamers using molecular dynamics and coarse-grained simulation methods. The multiscale character of the simulation is maintained by calculating thermal-average forces and diffusion coefficients of coarse-grained order parameters. Long-time simulations are performed for STMV capsid, HPV16 capsid, and individual pentamers belonging to these systems. The collapse transition of STMV capsid is investigated by starting from a partially collapsed structure. An individual pentamer of STMV is also studied for its stability in isolation. Conformational epitopes of HPV16 capsid protein are compared with those of the individual pentameric units. The additional stability of these epitopes arising from the complete icosahedral structure is studied. The role of curvature of the pentameric units in exposing epitope stabilizing residues is clarified. The higher immunogenicity of HPV16 VLPs compared to their L1 pentamers can be understood from the interactions between certain residues on the epitopes and the L1 pentamer.

Characterizing Changes In The Structure And Orientation Of Supported Model Membranes Upon Binding Of Cholera Toxin B

Cholera is caused by a protein toxin secreted by the bacterium Vibrio cholerae. This toxin is a member of the AB5 superfamily of toxins composed of an active (A) and a binding (B) pentameric unit. The five binding subunits of the pentamer bind specifically to the GM1, which is present in the outer leaflet of the host's plasma membrane; however, the mechanism for the translocation of the toxin is not well understood. Model membranes consisting of DMPC+cholesterol+GM1 were supported on gold electrode surfaces. The changes in the structure and orientation of the model membrane upon binding of the cholera toxin B unit were explored using differential capacitance, chronocoulometry and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Changes in the structure and orientation of the toxin protein with respect to a changing electric field were similarly investigated. The IR data suggests that the binding of the toxin to the membrane causes a decrease in the number of gauche conformers, which is caused by the constriction of the acyl chains due to interactions between the toxin and the GM1 glycolipids. The bound toxin induces some minor defects in the membrane; however, these defects are not significant enough to cause measurable changes in the average orientation of the membrane lipids. The major change in the bilayer upon binding of the cholera B unit was a remarkable decrease in the relative hydration of the membrane. This decrease in hydration is mostly like due to the separation of the bilayer from the aqueous electrolyte by the presence of the bound protein layer on the membrane surface. This work is part of ongoing study to understand the mechanism for translocation of the cholera toxin across the plasma membrane.