CONFORMATION OF THE SEBACYL BETA(1)LYS82-BETA(2)LYS82 CROSS-LINK IN T-STATE HUMAN HEMOGLOBIN

The crystal structure of human T state hemoglobin crosslinked with bis (3,5-dibromo-salicyl) sebacate has been determined at 1.9 Angstrom res olution, The final crystallographic R factor is 0.168 with root-mean-s quare deviations (RMSD) from ideal bond distance of 0.018 Angstrom. Th e 10-carbon sebacyl residue found in the beta cleft covalently links t he two beta Lys82 residues, The sebacyl residue assumes a zigzag confo rmation with cis amide bonds formed by the NZ atoms of beta Lys82's an d the sebacyl carbonyl oxygens, The atoms of the crosslink have an occ upancy factor of 1.0 with an average temperature factor for all atoms of 34 Angstrom(2), An RMSD of 0.27 for all CA's of the tetramer is obs erved when the crosslinked deoxyhemoglobin is compared with deoxyhemog lobin refined by using a similar protocol, 2HHD [Fronticelli et al, J. Biol. Chem. 269: 23965-23969, 1994], Thus, no significant perturbatio ns in the tertiary or quaternary structure are introduced by the prese nce of the sebacyl residue, However, the sebacyl residue does displace seven water molecules in the beta cleft and the conformations of the beta(1)Lys82 and beta(2)Lys82 are altered because of the crosslinking, The carbonyl oxygen that is part of the amide bond formed with the NZ of beta(2)Lys82 forms a hydrogen bond with side chain of beta(2)Asn13 9 that is in turn hydrogen-bonded to the side chain of beta(2)Arg104, A comparison of the observed conformation with that modeled [Bucci et al, Biochemistry 35:3418-3425, 1996] shows significant differences, Th e differences in the structures can be rationalized in terms of compen sating changes in the estimated free-energy balance, based on differen ces in exposed surface areas and the observed shift in the side-chain hydrogen-bonding pattern involving beta(2)Arg104, beta(2)Asn139, and t he associated sebacyl carbonyl group. (C) 1998 Wiley-Liss, Inc.