Assistant Professor
Department of Biophysics and Biophysical Chemistry
School of Medicine

herschel.wade@jhmi.edu

709 Hunterian
725 N. Wolfe Street
Baltimore, MD 21205

Office: 410-502-5629
Lab: 410-955-83077

Energetics and Mechanisms of Biological Switches
The regulatory properties of biomolecular switches are universally significant. Systems functioning as such are key players in cellular communication, regulation and signal transduction.  All life forms require the capabilities of these highly effective devices.  Moreover, they are exploited at all cellular levels where they play key roles in developmental, homeostatic and adaptive processes.  To little surprise, the high appreciation for the fine-tuned properties of biological switches crosses several platforms.  From the biomedical standpoint, connections to human diseases make these systems attractive targets for preventative, reversal and curative therapies.  For biochemists, their signal-integrative activities illuminate unique opportunities for investigations involving both internal and external control of cellular machinery. Beyond biology, switches provide benchmarks and paradigms for artificial devices constructed for a variety of technological usages. Design aspirations are also bolstered by nature’s vast signal-sensing repertoires linked to an equally large number of diverse downstream activities.  Without molecular insights regarding switch functions, opportunities involving pre-defined control of cellular and non-biological processes remain at best limited.  Details as such are still lacking despite numerous structural and biochemical characterizations studies, which have not descriptions complete enough to make meaningful connections between structure, energetics, mechanism and biological activities.

My group employs a battery of biophysical and biochemical techniques to gain insights regarding fundamental connections between switch energetics, structure and function. Of particular interest are several questions:  How do switch energetics translate to finely tuned signal-sensing/transmitting and regulatory capabilities?  What are the factors that control the energetic poise of switch systems?  What factors determine the relative stabilities of significantly populated allosteric states and allows limited access to nonproductive switch states?  How do the intrinsic properties of individual functional sites and cooperative site-site interactions contribute to switch energetics?  To address these issues, our attentions are directed at members of the MerR transcription factor (TF) family that exhibit important biological properties and, at the same time, distill essential features of increasingly complex systems.  Significantly, MerR-related TFs are poised such that experimental access is granted for all mechanistically relevant switch species.  Current experiments are formulated to (1) examine the utilization of ligand-binding free-energy with the use small-molecule effectors as probes of allostery energetics; (2) identify and examine elements that facilitate intra-domain cooperativity; (3) elucidate the determinants responsible for the stabilization of “ON” and “OFF” states and the energetic poise between the two forms.  Biophysical insights into switch energetics will be complemented with in vivo studies and genetic approaches involving the generation, isolation and characterization of variants showing “off-pathway” (i.e. altered signal-response) characteristics.

Basis of Ligand-binding Specificity and Promiscuity
Principles that govern molecular recognition among interacting biological systems are important in regards to every process that happens in a cell.  Well-behaved, highly selective systems that follow the lock-and key paradigm can be studied in great deal with well-established methods including, namely site-directed mutagenesis and the use of ligand analogs.  From these studies, insights regarding biological specificity have been invaluable to our understanding of biology, drug development and the development of tools for studying biological mechanisms.  However, highly selective systems now appear to be the exception rather than rule.  Recently, biological promiscuity has been brought to light and it is now apparent that NOT all macromolecules recognize biological partners at the level once thought.  Some of these systems are important and medically related including; G-protein coupled receptors (GPCRs), adaptor protein domains (PH, PB, SH3, PDZ, etc.), ion channels, immunoglobulins, T-cell receptors, calmodulin and cytokine receptors.  Such systems interact with, in some cases, many partners, but in all cases, their behaviors do not appear indiscriminant.  When biochemical methods like site-directed mutagenesis are employed to study broadly specific systems, they appear to fail.  From a structural point of view, binding sites with relaxed specificity requirements are clearly different from those that provide a lock for the key, suggesting that methods employed to study specific pockets may not be appropriate for those with diffuse “selectivity filters.”

Factors that govern specificity—promiscuity will be examined using an ensemble of ligand—receptor systems that span a specificity-affinity continuum. Through the comparative analyses of the structurally homologous protein-ligand pairs, factors that influence specificity will be illuminated as enhancing features will become enriched and diminishing ones will be “washed away,” thus providing deeper understanding of how the structural properties of ligand pockets relates to ligand-binding properties.  The ensemble will be derived from the BmrR transcription factor, which recognizes numerous structurally unrelated cationic lipophilic ligands and regulates the expression of a multidrug efflux pump.  A convergent molecular library will be generated using phage display and biopanning selective pressures designed to enforce increases in binding specificity and affinity.  The binding properties of the variants will be characterized using thermodynamic, kinetic, and structural methods. Some specific issues to be addressed include those regarding the basis for BmrR’s specificity for cationic ligands, relationships between pocket structure and specificity—promiscuity as well as functional—structural roles played by key pocket residues.

Selected Publications
Wade, H., S. Stayrook, and W.F. DeGrado. (2006) The structure of a designed diiron(III) protein: implications for cofactor stabilization and catalysis. Angew. Chem. Int. Ed. Engl. 45:4951-4954.

Wei, P.P., A.J. Skulan, H. Wade, W.F. DeGrado, and E.I. Solomon. (2005) Spectroscopic and computational studies of the de novo designed protein DF2t: correlation to the biferrous active site of Ribonucleotide Reductase and factors that affect O2 reactivity. J. Am. Chem. Soc. 127:16098-16101.

Maglio, O., F. Nastri, J. Calhoun, S. Lahr, H. Wade, V. Pavone, W.F. DeGrado, and A. Lombari. (2005) Artificial diiron proteins: solution characterization of four-helix bundles containing two distinct types of inter-helical loops. J. Biol. Inorg. Chem. 10:539-49.

Wade, H., S. Stayrook, and W.F. DeGrado. (2003) Structural analysis of a de novo designed metalloprotein. J. Inorg. Biochem. 96:246.

Wade, H., and T.S. Scanlan. (2003) A thermodynamic analysis of transition-state stabilization and transition-state analog binding. ChemBioChem. 4:537-540.

Wade, H.,* Di Costanzo, L.,* S. Geremia, L. Randaccio, V. Pavone, W.F. Grado, and A. Lombardi. (2001) Towards the de novo design of a catalytically active helix bundle: A substrate-accessible carboxylate-bridged dinuclear metal center. J. Am. Chem. Soc. 123:12749-12757.
*first two authors contributed equally to the work.

Wade, H., and T.S. Scanlan. (1999) Expression of binding energy on an antibody reaction coordinate. J. Am. Chem. Soc. 121:11935-11941.

Wade, H., and T.S. Scanlan. (1999) Remote binding energy in antibody catalysis: Studies of a catalytically un-optimized specificity pocket. J. Am. Chem. Soc. 121:1434-1443.