Solution Structures of RNAs and Proteins

Professor Pardi's primary research interests are in the area of biophysical chemistry and NMR spectroscopy. High-resolution multi-dimensional NMR experiments are used to probe the structure and dynamics of biomolecules in solution. A long-range goal is to understand the relationship between the structrures (and dynamics) of these molecules and their biological functions.

One current area of interest is structural studies of RNA enzymes (ribozymes). There are a large number of reactions for which RNA has been shown to be the biological catalyst. The hammerhead ribozyme is one of the most biochemically studied systems and has been shown to efficiently and catalytically cleave specific sites in target RNAs. In Professor Pardi's lab, multidimensional NMR experiments are being used to probe the structure of the hammerhead ribozyme in solution.

The primasry structural data dervied from the NMR experiments are proton-proton internuclear distances. This distance information is used to determine the secondary and tertiary structure of the hammerhead RNA domain. The structural information is then combined with kinetic, mechanistic, and biochemical properties of these ribozymes (being obtained by Dr. Uhlenbeck's group in the Department) to aid in the design of optimal RNA cleavage reagents.

The Pardi lab has developed methods for generating NMR quantities of isotopically (¹³C and/or ¹⁵N) labeled RNAs. The use of isotopic labels makes it possible to increase the dimensionality of the NMR experiments. The researchers recentky reported the first applications of three- and four-dimensional heteronuclear NMR to structural studies of nucleic acids. These techniques enormously facilitate the resonance assignment and structural analysis of RNA. The tremendous increase in resolution in these experiments allows detailed structure determinations of much larger RNAs than were previously possible.

The Pardi group is applying these new NMR techniques to a variety of biologically important RNA systems including several catalytic RNAs, a family of naturally-occurring and unusually stable hairpins containing tetranucleotide loops, tRNAs and tRNA precursors, and RNA inhibitors of various enzymatic proteins. In addition, a number of model RNA systems are being studied to better define the structural motifs that constitute the building blocks for RNA tertiary interactions/

Another area of interest is the determination of the solution structures of biologically active peptides or small proteins. Multidimensional NMR and computational methods are again used to generate these structures. Current projects include the family of antimicrobial peptides known as defensins, and neurologically active Conotoxin peptides. These defensins form part of the oxygen-independent mammalian defense system against a broad spectrum of microbes including bacteria, fungi, and enveloped viruses. The defenses vary greatly in their potency and range of activities. The Pardi group has determined the three-dimensional structure of several defensins by NMR spectroscopy and is searching for correlations between variations in their structure and their distinct biological activities. This family of peptides provides an ideal natural laboratory for studying the effects of substitution of individual amino acids on the folding, structure, dynamics, and biological activity of peptides in solution.