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Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

ABSTRACT: The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

SUBMITTER: Buczek P

PROVIDER: S-EPMC2953474 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

Buczek Pawel P Horvath Martin P MP

Journal of molecular biology 20060425 5

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration c ...[more]

PMID: 16678852

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Project description:Signalling from membrane receptors through heterotrimeric G-proteins (G alpha and G beta gamma) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defined mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Go alpha (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Go alpha with known G beta gamma-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of G alpha to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal alpha-helix of Go alpha with a proline point mutation at Arg-9 (R9P) does not affect interactions with G beta gamma on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or deletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Go alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Go alpha and mutants translated in vitro to liposomes indicates that Go alpha preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Go alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of G alpha subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for regulating signal transduction.

Dataset Information

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

Publications

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

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