Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization.

Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization.

Patch formation of a viral channel forming protein within a lipid membrane--Vpu of HIV-1.

Ion channels and their viral companions are defined by their quaternary structure. The individual sub-units have to assemble into homo- or hetero-oligomers. Using Vpu of HIV-1, a putative viral channel forming protein (VCP), as a test case, the formation of a quaternary structure is monitored using coarse grained molecular dynamics (CGMD) simulations. Full length Vpu is generated by combining the helical transmembrane domain (TMD) with the cytoplasmic domain derived from NMR spectroscopy. Patches of 2 to 6 as well as patches of 16 and 32 Vpu proteins, Vpu-WT, containing unphosphorylated serines 52 and 56 are used to study assembly dynamics. The same patches are simulated for the Vpu double mutant, Vpu-DD, in which the two serines 52 and 56 are replaced by aspartic acid. Serines 52 and 56 in Vpu-WT allow short lived contacts between the cytoplasmic domains. Dimer formation is the first step for long lasting assemblies and is induced by the EYR motif. Roll-over movements allow rearrangement within the dimer. Independent of the number of Vpu proteins, Vpu-DD prefers smaller aggregates than Vpu-WT. In the case of simulation of 4 Vpu-WT proteins a pore-like assembly is directly identified with the TMD Ser-23 pointing towards a putative central pore axis.

Viral channel proteins in intracellular protein-protein communication: Vpu of HIV-1, E5 of HPV16 and p7 of HCV.

Viral channel forming proteins are known for their capability to make the lipid membrane of the host cell and its subcellular compartments permeable to ions and small compounds. There is increasing evidence that some of the representatives of this class of proteins are also strongly interacting with host proteins and the effectiveness of this interaction seems to be high. Interaction of viral channel proteins with host factors has been proposed by bioinformatics approaches and has also been identified experimentally. An overview of the interactions with host proteins is given for Vpu from HIV-1, E5 from HPV-16 and p7 from HCV. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Assembling an ion channel: ORF 3a from SARS-CoV.

Protein 3a is a 274 amino acid polytopic channel protein with three putative transmembrane domains (TMDs) encoded by severe acute respiratory syndrome corona virus (SARS-CoV). Synthetic peptides corresponding to each of its three individual transmembrane domains (TMDs) are reconstituted into artificial lipid bilayers. Only TMD2 and TMD3 induce channel activity. Reconstitution of the peptides as TMD1 + TMD3 as well as TMD2 + TMD3 in a 1 : 1 mixture induces membrane activity for both mixtures. In a 1 : 1 : 1 mixture, channel like behavior is almost restored. Expression of full length 3a and reconstitution into artificial lipid bilayers reveal a weak cation selective (PK ≈ 2 PCl ) rectifying channel. In the presence of nonphysiological concentration of Ca-ions the channel develops channel activity.

Mechanism of function of viral channel proteins and implications for drug development.

Viral channel-forming proteins comprise a class of viral proteins which, similar to their host companions, are made to alter electrochemical or substrate gradients across lipid membranes. These proteins are active during all stages of the cellular life cycle of viruses. An increasing number of proteins are identified as channel proteins, but the precise role in the viral life cycle is yet unknown for the majority of them. This review presents an overview about these proteins with an emphasis on those with available structural information. A concept is introduced which aligns the transmembrane domains of viral channel proteins with those of host channels and toxins to give insights into the mechanism of function of the viral proteins from potential sequence identities. A summary of to date investigations on drugs targeting these proteins is given and discussed in respect of their mode of action in vivo.

Molecular dynamics simulations and conductance studies of the interaction of VP1 N-terminus from Polio virus and gp41 fusion peptide from HIV-1 with lipid membranes.

The icosahedral Polio virus capsid consists of 60 copies of each of the coat proteins VP1, VP2, VP3 and myristolyated VP4 (myrVP4). Catalyzed by the host cell receptor the Polio virus enters the host cell via externalization of myrVP4 and the N terminal part of VP1. There are several assumptions about the individual role of both of the proteins in the mechanism of membrane attachment and genome injection. We use the first 32 N terminal amino acids of VP1 and applied molecular dynamics simulations to assess its mechanism of function when attached and inserted into hydrated lipid membranes (POPC). Helical models are placed in various positions in regard to the lipid membrane to start with. As a comparison, the first 33 amino acids of the fusion peptide of gp41 of HIV-1 are simulated under identical conditions. Computational data support the idea that VP1 is not penetrating into the membrane to form a pore; it rather lays on the membrane surface and only perturbs the membrane. Furthermore, this idea is strengthened by channel recordings of both peptides showing irregular openings.

Ultrasensitive in situ label-free DNA detection using a GaN nanowire-based extended-gate field-effect-transistor sensor.

In this study, we have successfully demonstrated that a GaN nanowire (GaNNW) based extended-gate field-effect-transistor (EGFET) biosensor is capable of specific DNA sequence identification under label-free in situ conditions. Our approach shows excellent integration of the wide bandgap semiconducting nature of GaN, surface-sensitivity of the NW-structure, and high transducing performance of the EGFET-design. The simple sensor-architecture, by direct assembly of as-synthesized GaNNWs with a commercial FET device, can achieve an ultrahigh detection limit below attomolar level concentrations: about 3 orders of magnitude higher in resolution than that of other FET-based DNA-sensors. Comparative in situ studies on mismatches ("hotspot" mutations related to human p53 tumor-suppressor gene) and complementary targets reveal excellent selectivity and specificity of the sensor, even in the presence of noncomplementary DNA strands, suggesting the potential pragmatic application in complex clinical samples. In comparison with GaN thin film, NW-based EGFET exhibits excellent performance with about 2 orders higher sensitivity, over a wide detection range, 10(-19)-10(-6) M, reaching about a 6-orders lower detection limit. Investigations illustrate the unique and distinguished feature of nanomaterials. Detailed studies indicate a positive effect of energy band alignment at the biomaterials-semiconductor hybrid interface influencing the effective capacitance and carrier-mobility of the system.

Modulating the activity of the channel-forming segment of Vpr protein from HIV-1.

Viral protein of regulation (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) is a short auxiliary protein that is 96 amino acids in length. During the viral life cycle, Vpr is released into the blood serum and is able to enter cellular membranes of noninfected cells. In this study a short peptide, Vpr(55-83), was shown to exhibit ion-channel-like activity when reconstituted into (1) planar lipid bilayers and (2) lipid bilayers held at the tip of a glass pipette. The two set-ups led to differences in the oligomerization state of the peptide, which was reflected in differences in the conductance levels. Experiments under applied hydrostatic pressure affect the dynamics of the protein within the membrane.

Label-free dual sensing of DNA molecules using GaN nanowires.

We demonstrate a rationale for using GaN nanowires (GaNNWs) in label-free DNA-sensing using dual routes of electrochemical impedance spectroscopy (EIS) and photoluminescence (PL) measurements, employing a popular target DNA with anthrax lethal factor (LF) sequence. The in situ EIS reveals that both high surface area and surface band-bending in the nanowires, providing more binding sites and surface-enhanced charge transfer, respectively, are responsible for the enhanced sensitivity to surface-immobilized DNA molecules. The net electron-transfer resistance can be readily deconvoluted into two components because of the coexistence of two interfaces, GaN/DNA and DNA/electrolyte interfaces, in series. Interestingly, the former, decreasing with LF concentration (C(LF)), serves as a signature for the extent of hybridization, while the latter as a fingerprint for DNA modification. For PL-sensing, the band-edge emission of GaNNWs serves as a parameter for DNA modification, which quenches exponentially with C(LF) as the incident light is increasingly blocked from reaching the core nanowire by rapidly developing a UV-absorbing DNA sheath at high C(LF). Furthermore, successful application for detection of "hotspot" mutations, related to the human p53 tumor-suppressor gene, revealed excellent selectivity and specificity, down to picomolar concentration, even in the current unoptimized sensor design/condition, and in the presence of mutations and noncomplementary strands, suggesting the potential pragmatic application in complex clinical samples.

DNA-gold nanorod conjugates for remote control of localized gene expression by near infrared irradiation.

Gold nanorods were attached to the gene of enhanced green fluorescence protein (EGFP) for the remote control of gene expression in living cells. The UV-vis spectroscopy, electrophoresis, and transmission electron microscopy (TEM) were used to study the optical and structural properties of the EGFP DNA and gold nanorod (EGFP-GNR) conjugates before and after femto-second near-infrared (NIR) laser irradiation. Upon NIR irradiation, the gold nanorods of EGFP-GNR conjugates underwent shape transformation that resulted in the release of EGFP DNA. When EGFP-GNR conjugates were delivered to cultured HeLa cells, induced GFP expression was specifically observed in cells that were locally exposed to NIR irradiation. Our results demonstrate the feasibility of using gold nanorods and NIR irradiation as means of remote control of gene expression in specific cells. This approach has potential applications in biological and medical studies.