Development of protein based bioremediation and drugs for heavy metal toxicity

Structural studies were performed on several proteins of the bacterial detoxification system. These proteins are responsible for binding (MerP) and transport of heavy metals, including mercury, across membranes. The structural information obtained from NMR experiments provides insight into the selectivity and sequestration processes towards heavy metal toxins

NMR studies of membrane proteins

Nuclear magnetic resonance studies of membrane proteins yield valuable insights into their structure and topology. For example, the tilt angle and rotation of the helices in an ion channel can be determined by solid-state NMR spectroscopy in aligned lipid bilayers. Details about the structure of the protein in aligned phospholipids environments are immediately apparent from inspection of the SAMMY spectrum and the data can be further used for the determination of atomic resolution three-dimensional structures. SAR by NMR is a technique that is well suited for the field of membrane transporter proteins. The experiments on protein/phospholipid samples provide a unique insight into the interaction of drugs and the functional proteins.The advances required to transform solid-state NMR from a spectroscopic technique to a generally applicable method for determining molecular structures included multiple-pulse sequences, double-resonance methods, and separated local field spectroscopy. It also required improvements in instrumentation, especially the use of high-field magnets and efficient probes capable of high-power radio-frequency irradiations at high frequencies. The pace of development is accelerating and the local field is being utilized in an increasing number of ways in spectroscopic investigations of molecular structure and dynamics. Applications to many helical membrane proteins are underway and promise to add to our understanding of membrane proteins in health and disease.Chemistr

Phage-Induced Alignment of Membrane Proteins Enables the Measurement and Structural Analysis of Residual Dipolar Couplings with Dipolar Waves and lambda-Maps

At pH \u3e 6 added filamentous bacteriophage fd is compatible with many of the detergents used to solubilize membrane proteins for solution NMR studies of membrane proteins and, therefore, serves as an alignment media. In combination with strained polyacrylamide gel alignment, Dipolar Waves can be used to directly assess the secondary structure and a λ-map extracts the order tensors for de novo structure calculation of membrane proteins without distance restraints

Structural determination of virus protein U from HIV-1 by NMR in membrane environments

AbstractVirus protein U (Vpu) from HIV-1, a small membrane protein composed of a transmembrane helical domain and two α-helices in an amphipathic cytoplasmic domain, down modulates several cellular proteins, including CD4, BST-2/CD317/tetherin, NTB-A, and CCR7. The interactions of Vpu with these proteins interfere with the immune system and enhance the release of newly synthesized virus particles. It is essential to characterize the structure and dynamics of Vpu in order to understand the mechanisms of the protein–protein interactions, and potentially to discover antiviral drugs. In this article, we describe investigations of the cytoplasmic domain of Vpu as well as full-length Vpu by NMR spectroscopy. These studies are complementary to earlier analysis of the transmembrane domain of Vpu. The results suggest that the two helices in the cytoplasmic domain form a U-shape. The length of the inter-helical loop in the cytoplasmic domain and the orientation of the third helix vary with the lipid composition, which demonstrate that the C-terminal helix is relatively flexible, providing accessibility for interaction partners

High-Resolution Structures and Orientations of Antimicrobial Peptides Piscidin 1 and Piscidin 3 in Fluid Bilayers Reveal Tilting, Kinking, and Bilayer Immersion

While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of 1H–15N dipolar couplings and 15N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (∼25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ∼1.2–3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains

NMR studies of p7 protein from hepatitis C virus

The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10° and 25° relative to the bilayer normal. A truncated construct corresponding to the second transmembrane domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix segment tilted at 10° is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7

The Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers and Interactions with IL-8

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth(1-3). IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation(2). CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors(4-7). Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences(8), as well as addition of stabilizing antibodies and small molecules(9) that facilitate crystallization in cubic phase monoolein mixtures(10). The intracellular loops of GPCRs are critical for G-protein interactions(11) and activation of CXCR1 involves both N-terminal residues and extracellular loops(2,12,13). Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity(14). Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed

Structural Biology of The sequestration & Transport of Heavy Metal Toxins: NMR Structure Determination of Proteins Containing the CYS-X-Y-Metal Binding Motif

The support from the Department of Energy enabled us to initiate research on several proteins from the bacterial mercury detoxification system; in particular, we were able to determine the structures of MerP and related metal binding sequences. We have also worked on the membrane transport proteins MerF and MerT