Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex.

Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (VH) and variable light (VL) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, VH-VL M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8VL domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8VL forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8VL homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both VH-VL M8 and M8VL, led us to rationally design tandem, covalent homodimers of M8VL domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.

Specific 13C labeling of Thr methyl groups has been accomplished via the growth of a standard laboratory strain of Escherichia coli on [2-13C]glycerol in the presence of deuterated isoketovalerate, Ile, and Ala. Diversion of the label from the Thr biosynthetic pathway is suppressed by including Lys, Met, and Ile in the growth medium. This method complements the repertoire of methyl labeling schemes for NMR structural and dynamic studies of proteins and is particularly useful for the study of nucleic acid binding proteins because of the high propensity of Thr residues at protein–DNA and −RNA interfaces.