Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of similar to 70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand-and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.

Oxygen reduction and proton translocation by cytochrome c oxidase

Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.

Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein

The c-Fos–c-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions.

Calculations of the free energy of interaction of the c-Fos−c-Jun coiled coil: Effects of the solvation model and the inclusion of polarization effects

The leucine zipper region of activator protein-1 (AP-1) comprises the c-Jun and c-Fos proteins and constitutes a well-known coiled coil protein−protein interaction motif. We have used molecular dynamics (MD) simulations in conjunction with the molecular mechanics/Poisson−Boltzmann generalized-Born surface area [MM/PB(GB)SA] methods to predict the free energy of interaction of these proteins. In particular, the influence of the choice of solvation model, protein force field, and water potential on the stability and dynamic properties of the c-Fos−c-Jun complex were investigated. Use of the AMBER polarizable force field ff02 in combination with the polarizable POL3 water potential was found to result in increased stability of the c-Fos−c-Jun complex. MM/PB(GB)SA calculations revealed that MD simulations using the POL3 water potential give the lowest predicted free energies of interaction compared to other nonpolarizable water potentials. In addition, the calculated absolute free energy of binding was predicted to be closest to the experimental value using the MM/GBSA method with independent MD simulation trajectories using the POL3 water potential and the polarizable ff02 force field, while all other binding affinities were overestimated.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Theoretical Studies on Coupled Electron and Proton Transfer in Cytochrome c Oxidase

The complexity of life is based on an effective energy transduction machinery, which has evolved during the last 3.5 billion years. In aerobic life, the utilization of the high oxidizing potential of molecular oxygen powers this machinery. Oxygen is safely reduced by a membrane bound enzyme, cytochrome c oxidase (CcO), to produce an electrochemical proton gradient over the mitochondrial or bacterial membrane. This gradient is used for energy-requiring reactions such as synthesis of ATP by F0F1-ATPase and active transport. In this thesis, the molecular mechanism by which CcO couples the oxygen reduction chemistry to proton-pumping has been studied by theoretical computer simulations. By building both classical and quantum mechanical model systems based on the X-ray structure of CcO from Bos taurus, the dynamics and energetics of the system were studied in different intermediate states of the enzyme. As a result of this work, a mechanism was suggested by which CcO can prevent protons from leaking backwards in proton-pumping. The use and activation of two proton conducting channels were also enlightened together with a mechanism by which CcO sorts the chemical protons from pumped protons. The latter problem is referred to as the gating mechanism of CcO, and has remained a challenge in the bioenergetics field for more than three decades. Furthermore, a new method for deriving charge parameters for classical simulations of complex metalloenzymes was developed.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutL alpha which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX(2)EX(4)E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and it mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX(2)EX(4)E motif.

The Noether-Lefschetz theorem for the divisor class group

Let X be a normal projective threefold over a field of characteristic zero and vertical bar L vertical bar be a base-point free, ample linear system on X. Under suitable hypotheses on (X, vertical bar L vertical bar), we prove that for a very general member Y is an element of vertical bar L vertical bar, the restriction map on divisor class groups Cl(X) -> Cl(Y) is an isomorphism. In particular, we are able to recover the classical Noether-Lefschetz theorem, that a very general hypersurface X subset of P-C(3) of degree >= 4 has Pic(X) congruent to Z.

Dependence of pH and surfactant effect in the synthesis of magnetite(Fe3O4) nanoparticles and its properties

Nanoparticles of Fe3O4 were synthesized by co-precipitation in an aqueous solution containing ferrous and ferric salts (1:2) at varying pH with ammonia as a base. It was found that the value of pH influences the reaction mechanism for the formation of Fe3O4. Furthermore, the addition of mercaptoethanol significantly reduced the crystalline size of Fe3O4 nanoparticles from 15.03 to 8.02 nm. X-ray diffraction (XRD) spectra revealed that the synthesized nanoparticles were epsilon-Fe2O3 or Fe3O4 phase. To further prove the composition of the product, as-prepared Fe3O4 were examined by X-rayphotoelectron spectroscopy (XPS). Magnetic properties of the obtained particles were determined by vibrating sample magnetometer (VSM). Further analysis of the X-ray studies shows that while maintaining a pH value of 6 and 9 in a solution containing iron salts II and III ions produces epsilon-Fe2O3. Whereas a pH value of 11 produces magnetite (Fe3O4) phase. All of these results show that the pH has a major role in the observed phase formation of (Fe3O4) nanoparticles.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec: H1t is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

Mycobacterium tuberculosis FtsZ requires at least one arginine residue at the C-terminal end for polymerization in vitro

We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (MtFtsZ) have any role in MtFtsZ polymerization in vitro. MtFtsZ-delta C1, which lacks C-terminal extreme Arg residue (underlined in the C-terminal extreme stretch of 13 residues, DDDDVDVPPFMRR), but retaining the penultimate Arg residue (DDDDVDVPPFMR), polymerizes like full-length MtFtsZ in vitro. However, MtFtsZ-delta C2 that lacks both the Arg residues at the C-terminus (DDDDVDVPPFM), neither polymerizes at pH 6.5 nor forms even single- or double-stranded filaments at pH 7.7 in the presence of 10 mM CaCl2. Neither replacement of the penultimate Arg residue, in the C-terminal Arg deletion mutant DDDDVDVPPFMR, with Lys or His or Ala or Asp (DDDDVDVPPFMK/H/A/D) enabled polymerization. Although MtFtsZ-delta C2 showed secondary and tertiary structural changes, which might have affected polymerization, GTPase activity of MtFtsZ-delta C2 was comparable to that of MtFtsZ. These data suggest that MtFtsZ requires an Arg residue as the extreme C-terminal residue for polymerization in vitro. The polypeptide segment containing C-terminal 67 residues, whose coordinates were absent from MtFtsZ crystal structure, was modeled on tubulin and MtFtsZ dimers. Possibilities for the influence of the C-terminal Arg residues on the stability of the dimer and thereby on MtFtsZ polymerization have been discussed.