Charles G. Hoogstraten

Water counting: quantitating the hydration level of paramagnetic metal ions bound to nucleotides and nucleic acids.

Binding of divalent metal ions plays a key role in the structure and function of ribozymes and other RNAs. In turn, the energetics and kinetics of the specific binding process are dominated by the balance between the cost of dehydrating the aqueous ion and the energy gained from inner-sphere interactions with the macromolecule. In this work, we introduce the use of the pulsed EPR technique of 2H Electron Spin-Echo Envelope Modulation (ESEEM) to determine the hydration level of Mn2+ ions bound to nucleotides and nucleic acids. Mn2+ is an excellent structural and functional mimic for Mg2+, the most common divalent ion of physiological interest. Comparison of data in D2O and H2O, with aqueous Mn2+ as a reference standard, allows a robust and precise determination of the number of bound water molecules, and therefore the number of RNA-derived ligands. Examples of applications to the mononucleotide models MnGMP and MnATP, as well as to the paradigmatic RNA system tRNAPhe, are shown.

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion.

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton–aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton–ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.

Intermolecular domain docking in the hairpin ribozyme: Metal dependence, binding kinetics and catalysis

The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH3)63+. RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a “double conformational capture” model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

Unraveling the thermodynamics and kinetics of RNA assembly: Surface plasmon resonance, isothermal titration calorimetry, and circular dichroism

The mechanisms and driving forces of the assembly of RNA tertiary structure are a topic of much current interest. In several systems, including our own work in the docking transition of the hairpin ribozyme, intramolecular RNA tertiary folding has been converted into an intermolecular binding event, allowing the full power of contemporary biophysical techniques to be brought to bear on the analysis. We review the use of three such methods: circular dichroism to isolate the binding of multivalent cations coupled to tertiary assembly, surface plasmon resonance to determine the rates of association and dissociation, and isothermal titration calorimetry to dissect the thermodynamic contributions to RNA assembly events. We pay particular attention to practical aspects of these studies, such as careful preparation of samples with fixed free concentrations of cations in order to avoid errors due to ion depletion effects that are common in RNA systems. Examples of applications from our own work with the hairpin ribozyme are shown. Distinctions among the data handling procedures for the various techniques used and solution conditions encountered are also discussed.

Intrinsic Base-Pair Rearrangement in the Hairpin Ribozyme Directs RNA Conformational Sampling and Tertiary Interface Formation

Dynamic fluctuations in RNA structure enable conformational changes that are required for catalysis and recognition. In the hairpin ribozyme, the catalytically active structure is formed as an intricate tertiary interface between two RNA internal loops. Substantial alterations in the structure of each loop are observed upon interface formation, or docking. The very slow on-rate for this relatively tight interaction has led us to hypothesize a double conformational capture mechanism for RNA-RNA recognition. We used extensive molecular dynamics simulations to assess conformational sampling in the undocked form of the loop domain containing the scissile phosphate (loop A). We observed several major accessible conformations with distinctive patterns of hydrogen bonding and base stacking interactions in the active-site internal loop. Several important conformational features characteristic of the docked state were observed in well-populated substates, consistent with the kinetic sampling of docking-competent states by isolated loop A. Our observations suggest a hybrid or multistage binding mechanism, in which initial conformational selection of a docking-competent state is followed by induced-fit adjustment to an in-line, chemically reactive state only after formation of the initial complex with loop B.

Fighting through the darkness

During the fourth year of my Ph.D. program, I found myself walking through the door of my universitys counseling center, seeking help for a dangerously strong urge to commit suicide. With the help of the compassionate people in that office, I pulled through the immediate crisis. Long-term

Approaches to the Determination of More Accurate Cross-Relaxation Rates and the Effects of Improved Distance Constraints on Protein Solution Structures

In the past decade, nuclear magnetic resonance (NMR) has become an accepted and widely-used technique for studying the structure and dynamics of small- to moderate-sized proteins, nucleic acids, oligosaccharides, and molecular complexes (Wuthrich, 1986; Clore and Gronenborn, 1991). The fundamental phenomenon that allows the extraction of three-dimensional structural information from NMR studies is cross relaxation between protons (Neuhaus and Williamson, 1989). While other NMR parameters such as chemical shifts and J-coupling constants also contain structural information (Case et al., 1994), cross relaxation as manifested in the nuclear Overhauser effect (NOE) is unique in providing pairwise information: each cross peak in a two-dimensional NOESY (Nuclear Overhauser Effect SpectroscopY) spectrum provides evidence of a short-range through-space interaction between two identified protons, and a macromolecular NOESY dataset may contain thousands of such peaks. Typically, each assigned cross peak is used to derive a distance “constraint,” or pair of bounds between which the distance is assumed to lie, and the dataset of bounds (perhaps along with additional information) is fed into one of several complex computer algorithms for conversion to a set of three-dimensional coordinates. Thus, the accuracy and precision of distances derived from NOESY spectra, and the effect of such accuracy and precision on the derived macromolecular structures, are key considerations in NMR structure determinations. In this Chapter, we review the development of experimental and calculational NMR techniques that allow improved accuracy in the determination of cross-relaxation rates and assess the usefulness of these techniques in structure determinations in proteins and nucleic acids.