Linda Jen-Jacobson

Structural and thermodynamic strategies for site-specific DNA binding proteins.

BACKGROUND Site-specific protein-DNA complexes vary greatly in structural properties and in the thermodynamic strategy for achieving an appropriate binding free energy. A better understanding of the structural and energetic engineering principles might lead to rational methods for modification or design of such proteins. RESULTS A novel analysis of ten site-specific protein-DNA complexes reveals a striking correspondence between the degree of imposed DNA distortion and the thermodynamic parameters of each system. For complexes with relatively undistorted DNA, favorable enthalpy change drives unfavorable entropy change, whereas for complexes with highly distorted DNA, unfavorable DeltaH degrees is driven by favorable DeltaS degrees. We show for the first time that protein-DNA associations have isothermal enthalpy-entropy compensation, distinct from temperature-dependent compensation, so DeltaH degrees and DeltaS degrees do not vary independently. All complexes have favorable DeltaH degrees from direct protein-DNA recognition interactions and favorable DeltaS degrees from water release. Systems that strongly distort the DNA nevertheless have net unfavorable DeltaH degrees as the result of molecular strain, primarily associated with the base pair destacking. These systems have little coupled protein folding and the strained interface suffers less immobilization, so DeltaS degrees is net favorable. By contrast, systems with little DNA distortion have net favorable DeltaH degrees, which must be counterbalanced by net unfavorable DeltaS degrees, derived from loss of vibrational entropy (a result of isothermal enthalpy-entropy compensation) and from coupling between DNA binding and protein folding. CONCLUSIONS Isothermal enthalpy-entropy compensation implies that a structurally optimal, unstrained fit is achieved only at the cost of entropically unfavorable immobilization, whereas an enthalpically weaker, strained interface entails smaller entropic penalties.

ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity

The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu2+ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu2+ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu2+-ion based long-range distance constraints. Double electron-electron resonance data yield Cu2+-Cu2+ and Cu2+-nitroxide distances that are uniquely consistent with one Cu2+ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu2+ ions bind per complex. Unexpectedly, Mg2+-catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu2+ binding at these hitherto unknown sites, 13 Å away from the Mg2+ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu2+ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu2+, the Mg2+-dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable double-strand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg2+ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg2+. Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.

Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point

We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds lambda-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy, and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the methods precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.

Fluorescent marker for direct detection of specific dsDNA sequences

We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzymes binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the markers ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.

Specific Labeling of Threonine Methyl Groups for NMR Studies of Protein–Nucleic Acid Complexes

Specific (13)C labeling of Thr methyl groups has been accomplished via the growth of a standard laboratory strain of Escherichia coli on [2-(13)C]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.

The Energetic Contribution of Induced Electrostatic Asymmetry to DNA Bending by a Site-Specific Protein

DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic side chains that contact DNA phosphates and/or by replacement of a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes -2.3 kcal mol(-1) to -0.9 kcal mol(-1) (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of +1.3 kcal mol(-1). Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that the favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex.

Sensitive Cu2+–Cu2+ Distance Measurements in a Protein–DNA Complex by Double-Quantum Coherence ESR

Double quantum coherence (DQC) ESR spectroscopy is applied to measure the Cu(2+)-Cu(2+) distance in the EcoRI-DNA complex. A simple method is proposed to reduce the contribution of nuclear hyperfine and quadrupole interactions to such data. The effects of such interactions between the electron spin of Cu(2+) and neighboring nuclei on the DQC data make it difficult to measure the nanometer range interspin distance. The DQC data is in good agreement with results obtained by double electron electron resonance (DEER) spectroscopy. At the same time, the signal-to-noise ratio per shot in DQC is high. Taken together, these results provide impetus for further development of paramagnetic metal ion-based DQC techniques.

A trimeric DNA polymerase complex increases the native replication processivity

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

Simulating the dynamics and orientations of spin-labeled side chains in a protein-DNA complex.

Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin-labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed, and distance measurements were performed using the double electron electron resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. C(α)-C(α) distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.