Associate Professor
Department of Biology
Krieger School of Arts & Sciences

hill@jhu.edu

004 Mudd Hall
3400 N. Charles Street
Baltimore, MD 21218

Office: 410-516-6783
Lab: 410-516-6790

The long-term goals of our lab are to contribute to understanding how proteins fold and function. Our approach to this broad question is to design and engineer proteins followed by thermodynamic, kinetic, and structural characterization using a wide variety of biophysical techniques including heteronuclear, multidimensional NMR spectroscopy. We apply this approach to study the protein folding problem and also to study signal transduction systems with biomedical relevance.

Protein Design
Protein design involves an iterative process of three steps: design, synthesis, and characterization. The design step involves computer modeling to create a suitable amino acid sequence for a desired protein fold. The protein is then synthesized either biologically or chemically. Alternatively, a combination of both methods can be used to obtain the protein. Next, the thermodynamic, kinetic, structural, and dynamical properties of the protein are determined using circular dichroism spectroscopy, analytical ultracentrifugation, differential scanning calorimetry, fluorescence spectroscopy, and NMR spectroscopy. These properties of a protein are essential for understanding protein structure and function.

Protein Folding
Protein design can be used to gain insight into the protein folding problem. If we truly understand how the amino acid sequence of a protein folds and specifies its unique structure, then we should be able to design a protein from first principles and have it fold and specify the intended structure. Previously, an amino acid sequence had been designed to fold and specify a dimeric, four-helix bundle, a protein structural motif found throughout nature. We determined a high-resolution structure of this protein using NMR spectroscopy, which allowed a detailed investigation of how the designed sequence specified a unique structure. In addition, comparing the original protein to mutant proteins demonstrated that an interfacial hydrogen-bonding interaction is essential for specifying a unique structure. Further mutational analysis indicated that solvent-exposed residues of this protein, not just those in the hydrophobic core, are also essential for specifying a unique structure. We also determined that the loop residues play an active role in specifying a unique structure by creating and characterizing several proteins with entirely new loop regions. Thus, through an iterative process of design and rigorous characterization, the principles governing protein folding can be evaluated. We are now applying this approach to understand other ubiquitous protein structural motifs.

Signal Transduction
We can also apply the principles of protein design to understand signal transduction systems with significant biomedical relevance. Many of these systems involve a complex network of interactions between multifunctional proteins resulting in difficulties elucidating the key elements responsible for biological activity. By designing functional, minimalist versions of multifunctional soluble and membrane proteins, our group will identify the essential features involved in pathogenesis. These features can be incorporated into designed proteins for therapeutic uses with improved characteristics such as increased binding affinity, increased thermal stability, or increased solubility. Furthermore, this work will guide drug design of small molecules and peptidomimetics.

Selected Publications
Thuduppathy, G.R., and R.B. Hill. (2004) Applications of NMR spin relaxation methods for measuring biological motions. Methods Enzymol. 384:243-264.

Hill, R.B., C. Bracken, W.F. DeGrado, and A.G. Palmer III. (2000) Molecular motions and protein folding: Characterization of the backbone dynamics and folding equilibrium of alpha2D using 13C NMR spin relaxation. J. Am. Chem. Soc. 122:11610-11619.

Hill, R.B., D.P. Raleigh, A. Lombardi, and W.F. DeGrado. (2000) De novo design of helical bundles as models for understanding protein folding and function. Acc. Chem. Res. 33:745-754.

Salom, D., R.B. Hill, J.D. Lear, and W.F. DeGrado. (2000) Protons versus Amantadine: A competition for binding to the M2 tetrameric ion channel from Influenza A virus. Biochemistry 39:14160-14170.

Hill, R.B., J.-K. Hong, and W.F. DeGrado. (2000) Hydrogen bonded cluster can specify the native state of a protein. J. Am. Chem. Soc. 122:746-747.

Hill, R.B., and W.F. DeGrado. (2000) A polar, solvent-exposed residue can be essential for native protein structure. Structure 8:471-479.