An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.

The relative orientation and motions of domains within many proteins are key to the control of multivalent recognition, or the assembly of protein-based cellular machines. Current methods of structure determination have limited applicability to macromolecular assemblies, characterized by weak interactions between the constituents. Crystal structures of such complexes might be biased by packing forces comparable to the interdomain interactions, while the precision and accuracy of the conventional NMR structural approaches are necessarily limited by the restricted number of NOE contacts and by interdomain flexibility rendering the available NOE information uninterpretable. NMR relaxation studies are capable of providing "long-range" structural information on macromolecules in their native milieu. Here we determine directly the change in domain orientation between unligated and dual ligated subdomains of the SH(32) segment of Abelson kinase in solution, using the orientational dependence of nuclear spin relaxation. These results demonstrate that the change in domain orientation between unligated and ligated forms can be measured directly in solution.

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15 N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.

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