2D Saturation Transfer Difference NMR for Determination of Protein Binding Sites on RNA Guanine Quadruplexes.

Saturation transfer difference (STD) NMR is a technique that provides information on the intermolecular interfaces of heterogenous complexes by cross-saturation from one molecule to the other. In this case, selective saturation of protein protons is applied, and the cross-relaxation to the RNA sample results in a reduction of the peak intensities in the measured H1-H1 NOESY spectrum. This allows for a relatively rapid and simple method of identifying the protein binding interface of an RNA with assigned chemical shift data.

Insights into the RNA quadruplex binding specificity of DDX21.

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides.

Probing the kinetics and thermodynamics of copper(II) binding to Bacillus subtilis Sco, a protein involved in the assembly of the Cu(A) center of cytochrome c oxidase.

BsSco is a member of the Sco protein family involved in the assembly of the Cu(A) center within cytochrome c oxidase. BsSco forms a complex with Cu(II) that has properties consistent with dithiolate ligation. Stopped-flow UV-visible absorbance and fluorescence coupled with multiwavelength analysis reveal biphasic binding kinetics between BsSco and Cu(II). An initial species appears with absorbance centered at 382 nm at a copper concentration-dependent rate (2.9 x 10(4) M(-1) s(-1)). The initial species decays at a first-order rate (1.5 s(-1)) to the equilibrium form with a maximum at 352 nm. Formation of the BsSco-Cu(II) complex is accompanied by quenching of protein fluorescence. The copper concentration-dependent phase gives 70% of the total quenching, while the final 30% develops during the second phase of the absorbance change. The pH dependence of copper binding shows that the copper-dependent rate increases by 50-fold as the pH decreases from 8.5 to 5.5 with an apparent pK(a) of 6.7. The slower phase rate is independent of pH. Comparison of circular dichroism spectra between apo-BsSco and the BsSco-Cu(II) complex reveals a small change in the UV region consistent with a subtle conformational change upon copper binding. There is formation of a distinctive visible CD spectrum in the BsSco-Cu(II) complex. A model is presented in which the kinetic and thermodynamic stability of the BsSco-Cu(II) complex results from a two-step mechanism. Release of copper would be facilitated in the intermediate form of BsSco, and attaining such a low-Cu(II) affinity state may be important for BsSco's function in Cu(A) assembly.

Stability of oxidized, reduced and copper bound forms of Bacillus subtilis Sco.

Sco is an accessory protein required for assembly of the Cu(A) center of cytochrome c oxidase. Functions proposed for Sco include as a copper chaperone and as a thiol-disulfide exchange protein. Differential scanning calorimetry (DSC) is used here to assess the interaction between the Bacillis subtilis version of Sco (BsSco) and Cu(II). When BsSco binds Cu(II) its melting temperature increases by 23 degrees C, which corresponds to an equilibrium dissociation constant of 3.50 pM. In contrast BsSco exhibits a much weaker affinity for Cu(I) (K(D)=10 microM). BsSco-Cu(II) is stable over days indicating an extremely slow dissociation for BsSco-Cu(II). However, at high ionic strength in the presence of excess copper, BsSco-Cu(II) returns to its oxidized, disulfide-bonded state and loses its copper binding capacity with a half time of 100 s. DSC of BsSco at high ionic strength indicates an increase in stability of metal free, reduced BsSco combined with a small destabilization of BsSco-Cu(II). It is proposed that BsSco undergoes an ionic strength induced conformational change that promotes electron transfer from the thiol groups on BsSco to Cu(II) to effect copper release. Such a redox transformation could be an important aspect of the copper transfer role proposed for BsSco in Cu(A) assembly.