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Andrew L Feig

Publications and source records attributed to Andrew L Feig.

7 recordsLinked to original sources
PubMed2004

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation.

In proteins, empirical correlations have shown that changes in heat capacity (DeltaC(P)) scale linearly with the hydrophobic surface area buried upon folding. The influence of DeltaC(P) on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to DeltaC(P)s of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a DeltaC(P) of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated DeltaC(P) by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the DeltaC(P). We weigh the strengths and limitations of this method for determining DeltaC(P) values. Finally, we discuss the data in light of the physical origins of the DeltaC(P)s for RNA folding and consider their impact on biological function.

Base Sequence↗
PubMed2004

Salt-dependent heat capacity changes for RNA duplex formation.

Two short non-self-complementary RNA duplexes have been probed extensively to assess whether the heat capacity change (DeltaCP) for RNA duplex formation is sensitive to the solution ionic strength. Isothermal titration calorimetry data for duplex formation were collected as a function of temperature to obtain the DeltaCP values under each condition. Ionic strength was varied by repeating the measurements in the presence of 0.1-1.5 M added NaCl. In both cases, the DeltaCP for duplex formation was shown to have a significant dependence on the solution ionic strength, varying linearly with the log of the added NaCl. In each case, the DeltaCP became more negative at high ionic strength. Heat capacity changes have been much better studied in the area of protein folding where DeltaCP is not strongly dependent upon ionic strength but instead depends primarily on the burial of hydrophobic surface. Whereas hydrophobic burial is almost certainly involved in RNA duplex formation as well, the polyanionic nature of the backbone and the associated ion condensation effects are almost certainly a contributor to the observed ionic strength dependence of DeltaCPs for duplex formation. These data also hint at the possibility that nearest neighbor effects may be evident in the heat capacity changes associated with nucleic acid folding.

Base Sequence↗
PubMed2004

Entropy-driven folding of an RNA helical junction: an isothermal titration calorimetric analysis of the hammerhead ribozyme.

Helical junctions are extremely common motifs in naturally occurring RNAs, but little is known about the thermodynamics that drive their folding. Studies of junction folding face several challenges: non-two-state folding behavior, superposition of secondary and tertiary structural energetics, and drastically opposing enthalpic and entropic contributions to folding. Here we describe a thermodynamic dissection of the folding of the hammerhead ribozyme, a three-way RNA helical junction, by using isothermal titration calorimetry of bimolecular RNA constructs. By using this method, we show that tertiary folding of the hammerhead core occurs with a highly unfavorable enthalpy change, and is therefore entropically driven. Furthermore, the enthalpies and heat capacities of core folding are the same whether supported by monovalent or divalent ions. These properties appear to be general to the core sequence of bimolecular hammerhead constructs. We present a model for the ion-induced folding of the hammerhead core that is similar to those advanced for the folding of much larger RNAs, involving ion-induced collapse to a structured, non-native state accompanied by rearrangement of core residues to produce the native fold. In agreement with previous enzymological and structural studies, our thermodynamic data suggest that the hammerhead structure is stabilized in vitro predominantly by diffusely bound ions. Our approach addresses several significant challenges that accompany the study of junction folding, and should prove useful in defining the thermodynamic determinants of stability in these important RNA motifs.

Animals↗
PubMed2003

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure.

DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.

Bacterial Outer Membrane Proteins↗
PubMed2002

Cold denaturation of the hammerhead ribozyme.

Cold denaturation is a thermodynamic phenomenon resulting from a difference in the heat capacities, DeltaCp, of the folded and unfolded states of a macromolecule. Whereas this phenomenon has been extensively studied in proteins, it has been thought not to occur in nucleic acids due to a negligible DeltaCp of folding. Questioning the validity of this assumption, the low-temperature structure of the hammerhead ribozyme, a small catalytic RNA, was investigated by circular dichroism spectroscopy. In the presence of 10 mM Mg2+ at pH 5.0 and 40% methanol, a cold unfolding event likely corresponding to tertiary structure loss was observed with a Tm of -20 degrees C. In 500 mM NaCl at pH 6.6, and 40% methanol, large-scale unfolding of the ribozyme at both hot (Tm = 53 degrees C) and cold (Tm = -1 degrees C) temperatures occurred. Fitting of these data to a two-state model allowed determination of DeltaCp = 3.4 kJ mol-1 K-1, corresponding to >/=0.18 kJ K-1 (mol base pair)-1, in good agreement with recently published calorimetric values for DNA duplexes. These results constitute the first direct observation of cold denaturation of a nucleic acid, and point to the importance of DeltaCp terms in the thermodynamics of nucleic acid folding.

Base Sequence↗
PubMed2002

Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile.

Uridine-5'-diphospho-1-alpha-d-glucose (UDP-Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile. Fluorescent analogs of UDP-Glc have been prepared for use in our studies of the clostridial toxins. These compounds are related to the methylanthraniloyl-ATP compounds commonly used to probe the chemistry of ATP-dependent enzymes. The reaction of excess methylisatoic anhydride with UDP-Glc in aqueous solution yields primarily the 2' and 3' isomers of methylanthraniloyl-UDP-Glc (MUG). As the 2' and 3' isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods. TcdA binds MUG competitively with respect to UDP-Glc with an affinity of 15 +/- 2 microm in the absence of Mg2+. There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions. Using a competition assay, the affinity of UDP-Glc was determined to be 45+/-10 microm in the absence of Mg2+. The binding of UDP-Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90-600 microm, depending on the experimental conditions. These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme-substrate complex prior to initiation of the transferase chemistry.

Bacterial Toxins↗