Linker Loop Research Articles

An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC)

We present a high-definition structure of d(5mCCT3CCT3ACCT3CC), a DNA sequence which resembles a four-times repeat of the C-rich strand of telomeres and centromeres. The structure is monomeric. The CC stretches form four hemi-protonated C·C base-pairs, belonging to two parallel-stranded duplexes which intercalate head-to-tail into an i-motif core. The four grooves of the core are similar to those observed previously in i-motif tetramers, with P-P distances around 0.9 nm and 1.4 nm for the narrow and wide grooves, respectively. At 0°C, the structure is formed even at pH 7, despite the required protonation of cytidine pairs, suggesting that it may be biologically relevant.The intercalation topology of the i-motif core is read off the pattern of inter-residue cross-peaks along each groove: between H1′ protons across the narrow grooves, and between amino and H2′ protons across the wide grooves. In the hemi-protonated C·C pairs, the imino proton is shared equally between the two bases, as shown by the equal intensities of the NOESY cross-peaks between the imino proton and the two cis amino protons of the pair. Short inter-sugar distances and the direction of CH1′ bonds are consistent with CH1′⋯O4′ hydrogen bonds across the narrow grooves, as suggested by Berger et al. (1996). Proc. Natl. Acad. Sci. USA, 93, 12116–12121.At one extremity of the i-motif core, the T3A linker loops across one of the two wide grooves. It extends the core by stacking of A11, which also forms a strongly propeller-twisted reverse-Hoogsteen pair with T8. At the other extremity, the two T3linkers loop side by side across the two narrow grooves, extending the core by stacking of a T5·T16 pair which connects the two linkers. In this T·T pair between parallel strands, the hydrogen bonds are from imino proton to O4, and the base-pair lifetime is 6 ms at 0°C. The structures of segments 1 to 7 and 12 to 18, which form the i-motif core and the T3loops, are related by a 2-fold pseudo-symmetry: the geometries and environment are so similar that the NOESY spectra are barely resolved.These various interactions illustrate how linker sequences may affect the stability, intercalation topology and folding pattern of the intramolecular i-motif.

Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix.

We describe the thermodynamic properties of an intramolecular triple helix with two all-thymine linker loops in which the Hoogsteen strand is covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulfide bridge. We compare these properties to those of the corresponding intramolecular triplex without the disulfide crosslink. Optical and calorimetric measurements reveal that the uncrosslinked parent triplex melts in a biphasic manner above pH 6, with the initial triplex to duplex transition (Hoogsteen strand release) occurring at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogsteen transition such that the triplex and underlying hairpin duplex melt as a single transition under all conditions studied. Model independent thermodynamic data obtained by differential scanning calorimetry reveals the crosslink-induced increase in triplex thermal stability corresponds to a free energy stabilization of about 3 kcal/mol, with this stabilization being entirely entropic in origin. In other words, the crosslink is enthalpically neutral, but nevertheless, induces a triplex stabilization of 3 kcal/mol due to a reduction in the entropy change associated with triplex melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength dependence of the melting transitions. From a comparison of the melting transitions at different pH values and ionic strengths, we estimate that 0.4 more protons are associated with the crosslinked triplex state than with the uncrosslinked triplex, and 1.3 fewer counterions are released on melting the crosslinked triplex. We discuss how such crosslink-induced changes in proton binding and counterion release, in conjunction with potential changes in hydration and conformational freedom, could combine to give rise to the observed changes in entropy.

Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge.

TWIK-1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK-1 subunits can self-associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage-sensitive channels via a disulfide bridge.

Determination of the solution structure of apo calbindin D 9k by NMR spectroscopy

The three-dimensional structure of apo calbindin D9k has been determined using constraints generated from nuclear magnetic resonance spectroscopy. The family of solution structures was calculated using a combination of distance geometry, restrained molecular dynamics, and hybrid relaxation matrix analysis of the nuclear Overhauser effect (NOE) cross-peak intensities. Errors and inconsistencies in the input constraints were identified using complete relaxation matrix analyses based on the results of preliminary structure calculations. The final input data consisted of 994 NOE distance constraints and 122 dihedral constraints, aided by the stereospecific assignment of the resonances from 21 beta-methylene groups and seven isopropyl groups of leucine and valine residues. The resulting family of 33 structures contain no violation of the distance constraints greater than 0.17 A or of the dihedral angle constraints greater than 10 degrees. The structures consist of a well-defined, antiparallel four-helix bundle, with a short anti-parallel beta-interaction between the two unoccupied calcium-binding loops. The root-mean-square deviation from the mean structure of the backbone heavy-atoms for the well-defined helical residues is 0.55 A. The remainder of the ion-binding loops, the linker loop connecting the two sub-domains of the protein, and the N and C termini exhibit considerable disorder between different structures in the ensemble. A comparison with the structure of the (Ca2+)2 state indicates that the largest changes associated with ion-binding occur in the middle of helix IV and in the packing of helix III onto the remainder of the protein. The change in conformation of these helices is associated with a subtle reorganization of many residues in the hydrophobic core, including some side-chains that are up to 15 A from the ion-binding site.

Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.

The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant

The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.