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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. Memorandut¢ To: Dr. H. McAllister and Staff From: D.H.Ford Re: Site visit with Dr.D. W. Christianson, University of Philadelphia, PA, March 27, 1991. Grant No. 2782 entitled "X-ray studies of novel elastase- inhibitor complexes." Goals: As expressed by Dr. Christianson: 1). to evaluate the strength of noncovalent, intermolecular interactions of the carbon-halogen bond as they occur between specifically-halogen- ated inhibitors of elastase in solution; 2).to explorethe strucutnral chemistry of these interactions by examining enzyme-inhibitor complexes by X-ray crystallography; and 3). to create single crystals of a 1-antichymotrypsin for X-ray crystallographic structure determination. Once he understands the normal 3-dimensional structure as determined in this fashion, Dr. Christianson plans to utilize site-directed mutagenesis,to create inhibitors which are more active than the normal type in vivo. Observations: Dr. Christianson is a yo~ung, enthusiastic and obvio,usly well trained investigator, with what to me, appears to be an extrememely well equippe.d labaoratory. Further,-he has gathered about him a group of graduates students and fellows who are equally enthusiastic. Among his future studies is some collaboration with Dr. Jim Powers, in Atlanta.' Since I am clearly a neophyte in the field of X-ray diffraction, Dr. Christian~son presented a lucid overview of the subject, how the crystals are formed (or grown) and then a'nalyzed by either of his two diffraction apparatuses. The data is then processed by computer to provide finally a visual image of 3-dimensional structure. What I found extremely interesting was the degree to which a change in one amino acid in a protein would alter its total config- uration, while at the same time creating a homologue of the original protein which could*not be ditinguished by cytochemical means from the parent 'wild' type. If he is successful in creating 'a more efficient anti-prot-,.iase, Dr. Christianson plans, in co-operation-with Dr. Powers to engage in more biochemical enzymatic studies to test the effectiveness of his 'mutant' enzyme in preventing breakdown of elastin. Results: ri,AAC Dr. Christianson appears to have,excellent progress during the first 9 months of his program. All this is outlined in the progress report of his Rl application. Of some significance is that he has developed a technique for forming crystals of antichymotrypsin which only require 1-2 weeks rather than the previous method which required 6-7 months. He is also growing much larger crystals which permit a greater degree of resolution than heretofore.• CommenvTo me, X-ray diffraction studies always appeared somewhat esoEt.eric, which while useful in obtaining some idea of structure, were only tangentiav,ily useful in application to biologic systems (I display my ignorance). Dr. Christianson' presentation provided a

2 a real education as to how this approach may be incorporated into biological/biochemical• studies to great advantage. A great deal of attention is directed in most research today to homologies in amino acid sequences in proteins and peptides.... as if these-homolo'gies indicated a tremendous similarity in function between two or three peptides, etc. However, it is clear from the X-ray diffraction study of a molecule that even the change of one amino acid, or the incorporation of a halogen ion may,alter the organization of the molecule considerably. Thus, when one considers the nACh receptor in the mammalian CNS wherein there are 7.forms of the alpha peptide and 5 forms of the beta peptide, which indicate essentially only a change of. a few amino acids in each peptid.e chain, it is clear that the 'variant' receptor thus formed may have a vastly different structure and may react quite differently in relation to binding of its normal ligand (Acetylcholine or some other agonist, such as nicotine), or respond to some new agonist or antagonist. Briefly, however, I found this to be an exciting project, directed by a well informed investigator. Further, with the obvious value of such studies in relation to the protease/antiprotease relationship in emphysema, a-study which is of interest to the goals of CTR. DHF