Vira Tretyachenko-Ladokhina

Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor THE ROLE OF DNA DEFORMATION ENERGY

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.

Molecular mechanisms of functional rescue mediated by P53 tumor suppressor mutations

We have utilized both molecular dynamics simulations and solution biophysical measurements to investigate the rescue mechanism of mutation N235K, which plays a key role in the recently identified global suppressor motif of K235/Y239/R240 in the human p53 DNA-binding domain (DBD). Previous genetic analysis indicates that N235K alone rescues five out of six destabilized cancer mutants. However, the solution biophysical measurement shows that N235K generates only a slight increase to the stability of DBD, implying a rescue mechanism that is not a simple additive contribution to thermodynamic stability. Our molecular simulations show that the N235K substitution generates two non-native salt bridges with residues D186 and E198. We find that the nonnative salt bridges, D186-K235 and E198-K235, and a native salt bridge, E171-R249, are mutually exclusive, thus resulting in only a marginal increase in stability as compared to the wild type protein. When a destabilized V157F is paired with N235K, the native salt bridge E171-R249 is retained. In this context, the non-native salt bridges, D186-K235 and E198-K235, produce a net increase in stability as compared to V157F alone. A similar rescue mechanism may explain how N235K stabilize other highly unstable beta-sandwich cancer mutants.

Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworkers model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

Structure and Dynamics of the Cytidine Repressor DNA-Binding Domain

The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that differentially regulates nine operons. The natural operators in CytR-regulated promoters are comprised of a pair of degenerate recognition half-sites arranged as inverted repeats and separated by a variable length spacer. Spacers range from 0 to 9 basepairs accounting for up to a 25A translation and 310 degree rotation of the half-sites. Characterizing the interactions between the CytR DNA-binding domain (DBD) and DNA is critical to understanding the mechanism of differential gene regulation. Analysis of the DBD structure using NMR allowed us to assess both the structure and the dynamics of the DBD in relation to DNA sequence specificity. Here, we present the structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif similar to other proteins that interact with DNA. The structure of the DBD in the presence of recognition site DNA reveals a departure in helical orientation from other members of the LacR family. In addition, the DBD structure differs when bound to nonspecific DNA and populates two distinct conformations when free. Nonspecific binding results in measurable changes in protein dynamics when compared to the protein specifically bound to the udp half-site substrate. Thus for CytR, the transition from nonspecific association to specific recognition results in changes in protein mobility that are coupled to structural rearrangements.