Vince J. LiCata

Applications of Fluorescence Anisotropy to the Study of Protein-DNA Interactions

The use of fluorescence anisotropy to monitor protein-DNA interactions has been on the rise since its introduction by Heyduk and Lee in 1990. As a solution-based, true-equilibrium, real-time method, it has several advantages (and a few disadvantages) relative to the more classical methods of filter binding and the electrophoretic mobility shift assay (gel shift). This chapter discusses the basis for monitoring protein-DNA interactions using fluorescence anisotropy, as well as the advantages and disadvantages of the method, but the bulk of the chapter is devoted to experimental tips and guidance meant to augment existing reviews of the method. The focus is on the current primary use of the method: direct measurement of binding isotherms for protein-DNA interactions in vitro. A short summary of emerging applications of the method is also included.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins

Adipocyte lipid‐binding protein (ALBP) is one of a family of intracellular lipid‐binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three‐dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand‐binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein‐lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand‐specific changes. Comparable calculations with intestinal fatty‐acid‐binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end‐state structural distributions of the ligand entry portal. Proteins 33:577–589, 1998.

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.

Analysis of free energy versus temperature curves in protein folding and macromolecular interactions.

Plots of free energy versus temperature are commonly called stability curves or Gibbs-Helmholtz curves, and they have proven to be extremely useful in protein folding and ligand-binding studies. Curvature in a Gibbs-Helmholtz or stability plot is indicative of a heat capacity change, and some of their primary uses in biochemistry over the past few decades have included determining ΔCp values and comparing ΔCp values between two related processes. This chapter describes basic approaches for analyzing curved Gibbs-Helmholtz plots, along with two specific extensions of standard Gibbs-Helmholtz plot analysis: (1) translating ΔG of folding versus temperature into ΔH and ΔS versus temperature for comparing mesophilic-thermophilic protein pairs, and (2) fitting Gibbs-Helmholtz plots to determine if ΔCp changes with temperature or not. Neither of these extensions is new, but they are infrequently used, and their use is particularly germane to certain molecular interpretations of thermodynamic information from ΔG versus temperature curves. It is shown that translating ΔG of folding into ΔH and ΔS of folding versus temperature for a mesophilic-thermophilic protein pair can immediately influence possible structural hypotheses for thermal stabilization of thermophilic proteins. It is also shown that very small temperature-dependent heat capacity changes (ΔΔCp values) can be obtained from extended fits to ΔG versus temperature plots, and that these very small ΔΔCp values can have serious consequences for any attempt to correlate ΔCp with ΔASA for some reactions.

The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs‐Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔCp) of folding. If increased or enhanced non‐covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow–Klentaq homologous pair, the folding enthalpy (ΔHfold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaqs extreme free energy of folding (ΔGfold) originates from a significantly reduced entropic penalty of folding (ΔSfold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic–thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar “reduced ΔCp mechanism” for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced‐entropic‐penalty model. Proteins 2014; 82:785–793.

Thermodynamics of the DNA Structural Selectivity of the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the Delta C(p) of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.

Extreme free energy of stabilization of Taq DNA polymerase

We have examined the chemical denaturations of the Klentaq and Klenow large‐fragment domains of the Type 1 DNA polymerases from Thermus aquaticus (Klentaq) and Escherichia coli (Klenow) under identical solution conditions in order to directly compare the stabilization energetics of the two proteins. The high temperature stability of Taq DNA polymerase is common knowledge, and is the basis of its use in the polymerase chain reaction. This study, however, is aimed at understanding the thermodynamic basis for this high‐temperature stability. Chemical denaturations with guanidine hydrochloride report a folding free energy (ΔG) for Klentaq that is over 20 kcal/mol more favorable than that for Klenow under the conditions examined. This difference between the stabilization free energies of a homologous mesophilic–thermophilic protein pair is significantly larger than generally observed. This is due in part to the fact that the stabilization free energy for Klentaq polymerase, at 27.5 kcal/mol, is one of the largest ever determined for a monomeric protein. Large differences in the chemical midpoints of the unfolding (Cm) and the dependences of the unfolding free energy on denaturant concentration in the transition region (m‐value) between the two proteins are also observed. Measurements of the sedimentation coefficients of the two proteins in the native and denatured states report that both proteins approximately double in hydrodynamic size upon denaturation, but that Klentaq expands somewhat more than Klenow. Proteins 2004;00:000–000.

The Glutamate Effect on DNA Binding by Pol I DNA Polymerases: Osmotic Stress and the Effective Reversal of Salt Linkage

The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Deltan(ions)) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Deltan(ions) in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Deltan(ions). Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of approximately 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to approximately 150-200 waters released), suggesting that glutamate plays a significant osmotic role. Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.

Effects of Assembly and Mutations Outside the Active Site on the Functional pH Dependence of Escherichia coli Aspartate Transcarbamylase

Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M. P., Mallikarachchi, D., Matthew, J. B., and Allewell, N. M.(1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N. M.(1995) Proteins Struct. Funct. Genet., in press). To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different. In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of 1 pH unit. Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Schachman, H. K.(1992) Biochemistry 31, 6562-6569). The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the c1:c4 interface) and which is disrupted in the T R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the c:r subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 Å from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.

[28] Thermodynamic approaches to understanding aspartate transcarbamylase

Publisher Summary This chapter reviews the thermodynamic approaches that are used to investigate aspartate transcarbamylase (ATCase), the potential and the pitfalls of each, and the insights that have emerged. Analysis of the thermodynamics of ATCase has reached the point at which the driving forces for the various molecular interactions and conformational changes involved in the functional cycle are reasonably well defined. The first attempt to develop a thermodynamic model of the allosteric mechanism utilized the two-state model. Linkages between ionization reactions and ligand binding are detected in the first calorimetric experiments carried out with ATCase and are increasingly being recognized as central to catalysis and regulation. The thermal denaturation of ATCase and its subunits has been examined in detail by differential scanning calorimetry. Many lines of evidence indicate that electrostatic effects are central to catalysis and regulation of ATCase. In the case of ATCase, the thermodynamic parameters for the binding of a set of competitive inhibitors of carbamyl phosphate and L -Asp are evaluated by determining the enthalpy of binding by flow microcalorimetry at saturating ligand concentrations and determining the free energy of binding by spectrophotometric titration.