Our work is directed toward understanding biological processes at the molecular level, and putting  that knowledge to practical use. Frequently, we do this by designing and synthesizing probe molecules suited to particular problems. Redox-active metal chelates, photoaffinity probes, and radioactive metal chelates provide examples.

 

      Proteins and nucleic acids carry out specific functions, such as molecular recognition, information transfer, and catalysis.  These functions depend in detail on the structure of each macromolecule.  Experimental procedures such as affinity labeling, cross-linking, or partial digestion with enzymes are useful in identifying important structural features.  Likewise, cleavage of a protein by a reagent bound at a particular site can give information about the folding of the polypeptide chain.  It is preferred that the chain not be cleaved at the amino acid residue carrying the cleavage reagent, but rather at sites that are nearby in the three-dimensional structure (though possibly distant in the primary sequence).  We have achieved this by introducing a metal binding site at one position in a polypeptide chain.  We attached a "bifunctional" chelate (a derivative of EDTA containing iron) to a unique residue of the protein bovine serum albumin, treated the protein-chelate conjugate with H2O2 and ascorbate, and observed the production of three peptide fragments which together account for the entire polypeptide chain.  Remarkably, the reaction conditions do not destroy the amino acids at the site of cleavage; the sequences of the fragments exactly match that of the parent protein, with no gaps.  For standard sequencing procedures, the cleavage has the same result as cleavage by a proteolytic enzyme.  Isotope studies indicate that an oxygen atom is transferred from H2O2 to the new carboxyl terminus, consistent with nucleophilic attach by coordinated peroxide on peptide carbon.

 

       RNA polymerase is the enzyme that transcribes the information in genes (DNA) by catalyzing the synthesis of RNA.  We have prepared azide photoprobes that mimic the natural substrate of RNA polymerase, and trace the path of a growing RNA chain across the surface of the enzyme/DNA/RNA transcription complex.  The results provide a complete map of the proteins that the RNA contacts during its synthesis.  Bacterial RNA polymerase is a stable complex of five proteins, 2 alpha, beta, beta', and sigma.  The sigma protein confers the ability to specifically bind to a particular (promoter) sequence of DNA nucleotides, and begin RNA synthesis at exactly the right position.  There are several allowed promoter sequences, and we have found that sigma interacts with them in different ways.  We are now studying the specific interactions between sigma and the other subunits.

 

                 Monoclonal antibody technology allows the specificity of an antibody for its antigen to be used in targeting cancer cells.  Radiolabeled monoclonal antibodies have shown considerable promise for the early detection and therapy of cancer.  Many radionuclides of interest are metals (111In, 90Y, 68Ga, 99mTc, 67Cu, 57Co, etc.) and we have synthesized reagents and developed methods to stably attach these metal ions to monoclonal antibodies.  Chelators that can hold radiometals with high stability  under physiological conditions are essential to avoid excessive radiation damage to non-target cells.  We are further refining the technology by developing cleavable linkers between the metal chelate and the antibody.

 

               We employed a one-bead-one-peptide combinatorial library of fluorogenic extended peptide substrates for lysosomal proteolysis by the human liver enzymes cathepsin B and cathepsin D, and selected excellent candidates for further study.  the fluorescent bead methodology is sufficiently simple and straightforward to explore new alternatives even in highly specialized drug targeting contexts such as radioimmunotherapy.  Trials of monoclonal antibodies labeled with our reagents are now being carried out in several clinical research facilities.