Thermophoresis: The Case of Streptavidin and Biotin.

Thermophoretic behavior of a free protein changes upon ligand binding and gives access to information on the binding constants. The Soret effect has also been proven to be a promising tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behavior is sensitive to solute-solvent interactions. In this work, we perform systematic thermophoretic measurements of the protein streptavidin (STV) and of the complex STV with biotin (B) using thermal diffusion forced Rayleigh scattering (TDFRS).

Strong Adverse Contribution of Conformational Dynamics to Streptavidin-Biotin Binding.

Molecular dynamics plays an important role for the biological function of proteins. For protein ligand interactions, changes of conformational entropy of protein and hydration layer are relevant for the binding process. Quasielastic neutron scattering (QENS) was used to investigate differences in protein dynamics and conformational entropy of ligand-bound and ligand-free streptavidin.

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association.

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface.

Enhancing the Catalytic Deamination Activity of APOBEC3C Is Insufficient to Inhibit Vif-Deficient HIV-1.

The retroviral restriction factors of the APOBEC3 (A3) cytidine deaminase family catalyze the deamination of cytidines in single-stranded viral DNA. APOBEC3C (A3C) is a strong antiviral factor against viral infectivity factor (vif)-deficient simian immunodeficiency virus Δvif, which is, however, a weak inhibitor against human immunodeficiency virus (HIV)-1 for reasons unknown. The precise link between the antiretroviral effect of A3C and its catalytic activity is incompletely understood.

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis.

The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities.

Dengue virus NS4A cytoplasmic domain binding to liposomes is sensitive to membrane curvature.

Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1-48), has been proposed to contain an amphipathic α-helix (AH).

Recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4A for structural studies.

Background: Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening.

Full-length Vpu and human CD4(372-433) in phospholipid bilayers as seen by magic angle spinning NMR.

HIV-1 Vpu and CD4(372-433), a peptide comprising the transmembrane and cytoplasmic domain of human CD4, were recombinantly expressed in Escherichia coli, uniformly labeled with 13C and 15N isotopes, and separately reconstituted into phospholipid bilayers. Highly resolved dipolar cross-polarization (CP)-based solid-state NMR spectra of the two transmembrane proteins were recorded under magic angle sample spinning. Partial assignment of 13C resonances was achieved.

Structural features of antiviral DNA cytidine deaminases.

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans.

Mapping the interaction between the cytoplasmic domains of HIV-1 viral protein U and human CD4 with NMR spectroscopy.

Viral protein U (VpU) of HIV-1 plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. In this study, regions in the cytoplasmic domain of VpU involved in CD4 binding were identified by NMR spectroscopy.

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.

Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag.

Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

Background: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E.

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation.

NMR structural characterization of HIV-1 virus protein U cytoplasmic domain in the presence of dodecylphosphatidylcholine micelles.

The HIV-1 encoded virus protein U (VpU) is required for efficient viral release from human host cells and for induction of CD4 degradation in the endoplasmic reticulum. The cytoplasmic domain of the membrane protein VpU (VpUcyt) is essential for the latter activity. The structure and dynamics of VpUcyt were characterized in the presence of membrane simulating dodecylphosphatidylcholine (DPC) micelles by high-resolution liquid state NMR.

NMR structure of the transmembrane and cytoplasmic domains of human CD4 in micelles.

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4(+) cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes.

Integral membrane proteins in nanodiscs can be studied by solution NMR spectroscopy.

We present a two-dimensional solution NMR spectrum of an integral membrane protein (IMP) in a nanodisc. Solution NMR relies on rapid isotropic tumbling of the analyte with correlation times in the nanosecond range. IMPs in a cellular membrane do not satisfy this condition.

An indole-binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP.

The role of tryptophan as a key residue for ligand binding to the ubiquitin-like modifier GABA(A) receptor associated protein (GABARAP) was investigated. Two tryptophan-binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified.

Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins.

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport.

Structural consequences of phosphorylation of two serine residues in the cytoplasmic domain of HIV-1 VpU.

The human immunodeficiency virus type 1 (HIV-1) protein U (VpU) is an accessory protein responsible for enhancement of viral particle release and down regulation of the T-lymphocyte coreceptor CD4. Direct binding between the cytoplasmic domains of CD4 and VpU as well as phosphorylation of serines 53 and 57 in the cytoplasmic domain of VpU plays a central role in CD4 downregulation. We investigated structural consequences of phosphorylation of the two serines using nuclear magnetic resonance spectroscopy.

Structural characterization of the transmembrane and cytoplasmic domains of human CD4.

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains.

Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor.

The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction.

Solid-state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana.

Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335-365) contained the putative membrane anchor of the protein (residues 339-357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly (13)C- and (15)N-labelled residues (Phe 340, Val 350, Ala 364).

Lipid-ethanol interaction studied by NMR on bicelles.

The interaction of ethanol with phospholipids was studied in bicelles at a physiologically relevant ethanol concentration of 20 mM and a lipid content of 14 wt % by high-resolution NMR. Transient association of ethanol with magnetically aligned bicelles imparts a small degree of anisotropy to the solute. This anisotropy allows detection of residual (1)H-(1)H and (1)H-(13)C dipolar couplings, which are superimposed on scalar couplings.

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase.

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 degrees C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups.