diaz@aecom.YU.EDU (Dizzy Dan Diaz) (11/11/87)
The following briefly summarizes some of the highlights of the recent PROTEIN DOMAINS symposium held 2-3 November at the Waksman Institute for Microbiology at Rutgers University. SECOND DAY R. Bruce Merrifield Rockefeller University "The Development of Hormone Agonists Through Chemical Synthesis of Peptides" Merrifield's group is interested in designing glucagon agonists which bind the glucagon receptor but don't mediate the resultant biological activities (e.g., increased blood glucose). Unfortunately, much of the allotted time was spend discussing solid-phase synthesis, the development of which bought the speaker a Nobel a few years back. He discussed new methods of sidechain protection during synthesis and well as the use of light-sensitive attachments to the solid resin, making the removal of finished product easier. Kurt Wuthrich Institut fur Molekularbiologie und Biophysik, Switzerland "Three-dimensional Protein Structures in Solution as Viewed by Nuclear Magnetic Resonance" This was one of my favorite talks. I had heard that it was now possible, but I didn't know how well it worked. "It" refers to the determination of solution structures of macromolecules using NMR. Wuthrich discussed the three major kinds of NMR techniques used: Proton-proton NMR uses H-H connectivity to determine interatomic distances; COSY uses through-bond connectivity; NOESY (Nuclear Overhauser Enhancement Spectroscopy) uses through-space connectivity. A recent Science article reviews 2D NMR. Using their technique, Wuthrich and colleagues were able to detect errors in the published sequence of a protein they were working on. A problem is that 2 A, which represents the sum of Van der Waals radii of the atoms in question, is the present limit of resolution. The NMR and crystal structure of the protein tendamistat (sp?) were compared. The alpha-carbon skeleton structures were almost identical. Sidechain conformations were the biggest difference between the two, especially with sidechains near the surface of the protein. Right now the technique is most useful for small proteins <= 10 kD. Russell Doolitte UCSD "Protein Evolution" A domain, Doolittle reminded us, used to be defined as an independently folding module in a protein. The definition has been blurred as molecular biologists have continued to bastardize terms from other areas of biology to describe phenomena in their own domain :). The determination of the function of protein domains is made puzzling by the apparent ability to delete entire regions of proteins without known biological effect. The gene for clotting factor VII has an intronless region coding for ~1000 amino acid residues. When this huge region is deleted, there is no effect on biological activity. While many computer-armed molecular biologists (those rogues!) claim great significance for their searches for sequence similarity (not homology, Doolittle emphatically emphasizes), the way to determine if an alignment is truly significant is to scramble the "similar" sequence and recheck the alignment. Such searches for similarity can be predictive, as was the finding that the E. coli uvrA protein had a region similar to the Zn-binding fingers of certain eukaryotic gene regulatory factors. When they checked out uvrA protein what did they find? 2 moles of bound Zn per mol of protein. As for the great similarity vs homology debate caused by massive abuse of the second term, Doolittle says that is two proteins are >100 aa long and >25% identical, then it would be strong evidence for homology. Howard Nash NIMH "Biochemistry of Site-Specific Recombination: Structure and Function of a Multiprotein Complex" Nash studies the integration of lambda phage as a model of recombination. The 20-25 nt att site in E. coli is identical to a stretch on the lambda chromosome, the site of crossover. Int protein binds to both sequences; it is a site-specific topoisomerase. Integration host factor is a heterodimeric E. coli protein which forms part of the integration complex, the intasome. The sequence of IHF is similar to that of HU protein, a histone-like protein in E. coli. The structure of HU has been determined and by comparison, they have theorized that IHF binds DNA in the minor groove. This result is difficult to rationalize on the basis of present models of DNA-protein interactions. The problem is that if one assumes only H-bonding for specific binding, it is difficult to see how the available H-bond contacts in the minor groove could foster such specificity. -- dn/dx Dept Molecular Biology diaz@aecom.yu.edu Dan Diaz Albert Finkelstein College of Medicine