Abhishek Mazumder

Recognition of different DNA sequences by a DNA-binding protein alters protein dynamics differentially

λ‐Repressor–operator sites interaction, particularly OR1 and OR2, is a key component of the λ‐genetic switch. FRET from the dansyl bound to the C‐terminal domain of the protein, to the intercalated EtBr in the operator DNA indicates that the structure of the protein is more compact in the OR2 complex than in the OR1 complex. Fluorescence anisotropy reveals enhanced flexibility of the C‐terminal domain of the repressor at fast timescales after complex formation with OR1. In contrast, OR2 bound repressor shows no significant enhancement of protein dynamics at these timescales. These differences are shown to be important for correct protein–protein interactions. Altered protein dynamics upon specific DNA sequence recognition may play important roles in assembly of regulatory proteins at the correct positions.

A Synthetic Peptide Mimic of λ-Cro shows Sequence-Specific Binding in Vitro and in Vivo

Development of small synthetic transcription factors is important for future cellular engineering and therapeutics. This article describes the chemical synthesis of α-amino-isobutyric acid (Aib) substituted, conformationally constrained, helical peptide mimics of Cro protein from bacteriophage λ that encompasses the DNA recognition elements. The Aib substituted constrained helical peptide monomer shows a moderately reduced dissociation constant compared to the corresponding unsubstituted wild type peptide. A suitably cross-linked dimeric version of the peptide, mimicking the dimeric protein, recapitulates some of the important features of Cro. It binds to the operator site O(R)3, a high affinity Cro binding site in the λ genome, with good affinity and single base-pair discrimination specificity. A dimeric version of an even shorter peptide mimic spanning only the recognition helix of the helix-turn-helix motif of the Cro protein was created following the same design principles. This dimeric peptide binds to O(R)3 with affinity greater than that of the longer version. Chemical shift perturbation experiments show that the binding mode of this peptide dimer to the cognate operator site sequence is similar to the wild type Cro protein. A Green Fluorescent Protein based reporter assay in vivo reveals that the peptide dimer binds the operator site sequences with considerable selectivity and inhibits gene expression. Peptide mimics designed in this way may provide a future framework for creating effective synthetic transcription factors.

Ultrafast interfacial solvation dynamics in specific protein DNA recognition

An overwhelming number of structural and functional studies on specific protein-DNA complexes reveal the existence of water molecules at the interaction interface. What role does the interfacial water molecules play in determining the specificity of association is thus a critical question. Herein, we have explored the dynamical role of minor groove water molecules and DNA side chain flexibility in lambda repressor-operator DNA interaction using well-characterized DNA minor groove binder dye, Hoechst 33258. The most striking finding of our studies reveals that the solvation time scale corresponding to the minor groove water molecules (∼50 ps) and DNA side chain flexibility (∼10 ns) remain unaltered even in protein-DNA complex in comparison to unbound operator DNA. The temperature dependent study further reveals the slower exchange of minor grove water molecules with bulk water in DNA-protein complex in comparison to the unbound DNA. Detailed structural studies including circular dichroism (CD) and Förster resonance energy transfer (FRET) have also been performed to elucidate the interaction between protein and DNA.

Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

A Genetic Network That Balances Two Outcomes Utilizes Asymmetric Recognition of Operator Sites

Stability and induction of the lysogenic state of bacteriophage λ are balanced by a complex regulatory network. A key feature of this network is the mutually exclusive cooperative binding of a repressor dimer (CI) to one of two pairs of binding sites, O(R)1-O(R)2 or O(R)2-O(R)3. The structural features that underpin the mutually exclusive binding mode are not well understood. Recent studies have demonstrated that CI is an asymmetric dimer. The functional importance of the asymmetry is not fully clear. Due to the asymmetric nature of the CI dimer as well as its binding sites, there are two possible bound orientations. By fluorescence resonance energy transfer measurements we showed that CI prefers one bound orientation. We also demonstrated that the relative configuration of the binding sites is important for CI dimer-dimer interactions and consequent cooperative binding. We proposed that the operator configuration dictates the orientations of the bound CI molecules, which in turn dictates CI cooperative interaction between the O(R)1-O(R)2 or O(R)2-O(R)3, but not both. Modeling suggests that the relative orientation of the C- and N-terminal domains may play an important role in the mutually exclusive nature of the cooperative binding. This work correlates unique structural features of a transcription regulatory protein with the functional properties of a gene regulatory network.