Research
What to expect in the Laboratory:
Students in the Showalter Group will learn and apply state of the art NMR techniques. Most modern bio-NMR techniques are derived from the heteronuclear correlation experiment (left). Analysis is usually built around standard data fitting and statistical methods also applicable to a wide range of other biophysical and bioanalytical methods. See below for recent examples of research done in our lab.
NMR experiments used routinely in our laboratory include:
- Backbone and sidechain spin relaxation and dispersion: Used to quantify the conformational dynamics of biomolecules.
- Residual Dipolar Coupling (RDC) measurements: Used primarily as a structure determination or comparison tool.
- NMR titration experiments: Both titrations of ligand into macromolecule, which can be used to evaluate binding constants for weak interactions; and pH or other solution modifications.
Rather than focusing solely on NMR spectroscopy, our laboratory strives to take an interdisciplinary approach to biophysical chemistry. Two methods which powerfully compliment NMR spectroscopy are biomolecular calorimetry and molecular dynamics simulations. Our laboratory uses isothermal titration calorimetry (ITC) as the primary tool for quantitative binding constant determination and other thermodynamic characterization of events involving the molecules we study. Molecular dynamics simulations (MD) offer unique insights into the conformational states accessed by biomolecules on the sub-microsecond timescale and can be utilized in conjunction with NMR spin relaxation or RDC measurements to provide unparalleled mechanistic detail.
Recent News
The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs
In this paper we continue our work aiming to characterize the role of double-stranded RNA binding domains in microRNA processing, but now we switch focus to the second cleavage step with preliminary studies of Dicer. This work combines in vitro binding assays with NMR spectroscopy to document Dicer-dsRBDs RNA binding activity. As this study appeared in PLoS One, it is an open access publication and anybody is free to download the origina article from the publisher for fair use.
Please follow this external link for access to the article.
Carbon-Detected 15N NMR Spin Relaxation of an Intrinsically Disordered Protein: FCP1 Dynamics Unbound and in Complex with RAP74
We present two 13C-direct detection experiments for the measurement of 15N NMR spin relaxation that quantify backbone dynamics on a per-residue basis for IDPs in solution. These experiments have been applied to the intrinsically disordered C-terminal of FCP1. The results provide evidence that most of FCP1 remains highly dynamic in both states, while the 20 residues forming direct contact with RAP74 become more ordered in the complex.
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Atomistic Simulations Reveal Structural Disorder in the RAP74-FCP1 Complex
We have recently completed atomically detailed molecular dynamics simulations characterizing the interaction of the RAP74 Winged helix domain with the intrinsically disordered C-terminal of FCP1. These simulations illustrate the importance of hydrophobic contacts for stabilizing disordered protein complexes and provide new insight inot the mechanism of protein binding by winged helix domains. In conjunction with our recent NMR experiments identifying residual structure in unbound FCP1 (below), these simulations suggest that FCP1 loses relatively little conformational entropy upon binding and that the associated coupled folding-binding transition may be less sharp than expected.
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The C-terminus of FCP1 is Partially Helical in the Unbound State.
The C-terminus of FCP1 is intrinsically disordered in solution and adopts an alpha helical conformation upon binding to RAP74. We have recently shown by a combination of carbon-detected NMR and circular dichroism spectroscopy that the core of this binding region is partially helical in the unbound state and that the extent of helicity can be controlled through the addition of osmolytes and chaotropes.
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Differential pri-miRNA Binding Affinity in DGCR8-dsRBD1 and Drosha-dsRBD
MicroRNAs affect gene regulation by base pairing with mRNA and contribute to the control of cellular homeostasis. The first step in maturing primary-miRNA transcripts involves the enzyme Drosha and its co-factor DGCR8. Here we show through electrophoretic binding assays, NMR spectroscopy, and MD simulations that the double-stranded RNA binding domain of Drosha, which does not bind pri-miRNA on its own, has fundamentally different backbone dynamics from the high-affinity binding domain 1 of DGCR8.
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Simulations of the DGCR8 Core
Over the past decade, miRNAs have been shown to affect gene regulation by base pairing with messenger RNA and their misregulation has been directly linked with cancer. DGCR8, a protein containing two dsRNA binding domains in tandem, is vital for nuclear maturation of
primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. We have recently completed a molecular dynamics simulation study of the DGCR8 "Core" protein that reveals correlated dynamics responsible for changing the separation distance and orientation of the two dsRNA binding domains in a way that will facilitate interaction with structurally diverse miRNAs.
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Carbon-detected NMR of Intrinsically
Disordered Proteins
Intrinsically disordered proteins have emerged as critical components of cellular systems, contributing to cell signalling and disease. We seek to implement new NMR-based methodologies for quantifying the structure and dynamics of IDPs with the same level of rigor currently attainable for co-operatively folded proteins. Recently, we have published a pair of carbon detected NMR experiments that facilitate complete aliphatic 1H resonance assignment in IDPs. These experiments have been used along with previously published "protonless" NMR experiments to fully assign the resonances of the intrinsically disordered C-terminus of human FCP1.
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Please return soon if you would like to see more specific details regarding ongoing projects in the laboratory.