Composite Dipolar Recoupling: Anisotropy Compensated Coherence Transfer in Solid-State NMR

The efficiency of dipole-dipole coupling driven coherence transfer experiments in solid-state NMR spectroscopy of powder samples is limited by dispersion of the orientation of the internuclear vectors relative to the external magnetic field. Here we introduce general design principles and resulting pulse sequences that approach full polarization transfer efficiency for all crystallite orientations in a powder in magic-angle-spinning experiments. The methods compensate for the defocusing of coherence due to orientation dependent dipolar coupling interactions and inhomogeneous radio-frequency fields. The compensation scheme is very simple to implement as a scaffold (comb) of compensating pulses in which the pulse sequence to be improved may be inserted. The degree of compensation can be adjusted and should be balanced as a compromise between efficiency and length of the overall pulse sequence. We show by numerical and experimental data that the presented compensation protocol significantly improves the efficiency of known dipolar recoupling solid-state NMR experiment

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 Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane

Viruses: incredible nanomachines. New advances with filamentous phages

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account

Orientation and dynamics of transmembrane peptides: the power of simple models

In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function

Structural biology of the sequestration and transport of heavy metal toxins: NMR structure determination of proteins containing the -Cys-X-Y-Cys-metal binding motifs. 1997 annual progress report

'There are enormous amounts of heavy metals in the environment, much of it in the form of organometallic compounds resulting from various types of industrial and military waste. Nearly all of these metals and compounds are highly toxic to biological organisms including humans. However, some bacteria thrive in the presence of high concentrations of heavy metal toxins because they possess efficient mechanisms for the detoxification of these metals and compounds. Heavy metals appear to be universally toxic because of their non-selective chemistry, for example Hg(II) reacts with essentially all exposed sulfhydryl groups on proteins, thus, it may seem surprising that any organism at all can survive these chemical insults much less those that grow in a toxic milieu. However, the prebiotic environment was undoubtedly heavily polluted with heavy metals from geological processes, and the most primitive organisms simply had to evolve mechanisms for dealing with them if they were going to be able to utilize Cys, His, and the other amino acids that contribute to metal binding sites in their proteins. Genes associated with bacterial resistance to Ag, AsO{sub 2}, AsO{sub 4}, Bi, Cd, Co, CrO{sub 4}, Cu, Hg, iNi, TeO{sub 3}, TI, Pb, Zn, and other metals of environmental concern have been described (Silver, 1992; Silver and Walderhaug, 1995).

Structural biology of the sequestration and transport of heavy metal toxins: NMR structure determination of roteins containing the -Cys-X-Y-Cys-metal binding motifs. 1998 annual progress report

'The overall goal of the research is to apply the methods of structural biology, which have been previously used primarily in biomedical applications, to bioremediation. The authors are doing this by using NMR spectroscopy to determine the structures of proteins involved in the bacterial mercury detoxification system. The research is based on the premise that the proteins encoded in the genes of the bacterial detoxification system are an untapped source of reagents and, more fundamentally, chemical strategies that can be used to remove heavy metal toxins from the environment. The initial goals are to determine the structures of the proteins of the bacterial mercury detoxification systems responsible for the sequestration and transport of the Hg(II) ions in to the cell where reduction to Hg(O) occurs. These proteins are meP, which is water soluble and can be investigated with multidimensional solution NMR methods, and merT, the transport protein in the membrane that requires solid-state NMR methods. As of June 1998, this report summarizes work after about one and half years of the three-year award. The authors have made significant accomplishments in three aspects of the NMR studies of the proteins of the bacterial mercury detoxification system.