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PubMedDate not supplied

On the formation and crystallization of sickle hemoglobin macrofibers.

We have characterized new aspects of macrofiber structure and assembly which provide a mechanism for macrofiber formation from fibers. After the formation of fibers, HbS forms macrofibers by the association of small, organized bundles of partially fused fibers. These macrofibers consist of double strands, packed into antiparallel rows, and are identical to double strands found in crystalline HbS, except that the double strands in macrofibers are axially displaced from their crystalline position and are twisted about the particle axis, whereas in crystals they are linear. In lateral views, electron micrographs of macrofibers show prominent sets of "rows." We use the number of these rows to designate a particular type of macrofiber. In this study we present micrographs of macrofibers with 3 to 11 rows visible in lateral views. Such particles contain from 20 to 200 double strands. The pitch of a macrofiber is coupled to the number of rows in a manner so that the angle between the molecules in the outermost double strand is always 1.8 degrees. This observation has led us to propose that the factor limiting the extent of lateral growth of macrofibers is distortions in bonding between the hemoglobin molecules in the outermost double strands. Similar considerations have provided an explanation of the factors that limit the lateral growth of fibers. Finally, we propose a simple mechanism for the formation of macrofibers from fibers. This mechanism postulates that integral numbers of fibers form specific types of macrofibers and has the virtue of conserving the polarity of the fibers.

Anemia, Sickle Cell↗

PubMedDate not supplied

On the stability of sickle hemoglobin macrofibers: isolated layers of antiparallel double strands.

Sickle hemoglobin macrofibers consist of rows of antiparallel double strands twisted about the particle axis. Under appropriate conditions the outer two rows can dissociate from the particle. These structures retain the helical twist of the parent macrofiber and slowly dissociate to monomers over a period of several hours. Individual double strands or pairs of parallel double strands are never observed, suggesting that only the antiparallel pairing is energetically favorable.

Crystallization↗

PubMedDate not supplied

Analysis of the intermolecular contacts within sickle hemoglobin fibers: effect of site-specific substitutions, fiber pitch, and double-strand disorder.

An atomic model of the sickle hemoglobin (HbS) fiber was synthesized by combining the molecular coordinates of the fiber (obtained from electron microscopy) with atomic coordinates of the sickle hemoglobin double strand (obtained from X-ray crystallography). The model is stereochemically acceptable. The majority of polymerization-sensitive HbS mutants are located at fiber contact sites and the majority of the mutants that do not affect polymerization are not located at contact sites. The residues at intermolecular contacts in the fiber model are reported. We have searched the coordinate space in the vicinity of the EM reconstructions to find models with alternative sets of coordinates that satisfy the mutant data, contain 5-A contacts between double strands, and are stereochemically acceptable. This involved a systematic examination over 297 different models. The alternative fiber models were generated with a range of fiber pitch, double-strand positions, and double-strand polarity. Models which had unacceptably close contacts between atoms, failed to satisfy the mutant data, or did not have 5-A contacts between double strands were considered unacceptable. None of the acceptable alternative fiber models improved the agreement between the polymerization behavior of HbS mutants and their contact site location. However, several models could account for the polymerization data equally well. Residue locations for single-site HbS mutations that could discriminate between alternative fiber models are proposed. The twist of HbS fibers varies in an apparent random manner with an average rotation of 7.8 +/- 2.5 degrees per molecule and a maximum rotation of 16 degrees per molecule. The number of interdouble-strand contacts as a function of fiber twist shows a broad maximum around 9 degrees and may account for the observed range of fiber pitch. This study shows that the upper limit on the fiber twist could result from a loss of axial contacts and repulsive van der Waals interactions between residues involved in interstrand contacts. The loss of axial contacts limits the radial growth of the fiber. In the appendix we analyze the methodology used by I. Cretegny and S. J. Edelstein [(1993) J. Mol. Biol. 230, 733-738] to build a model of the fiber. Our examination reveals shortcomings in the methodology of Cretegny and Edelstein. One result of these shortcomings is that the model synthesized by Cretegny and Edelstein is not stereochemically acceptable because it gives rise to a large number of excessively close (less than 1.4 A) atom-atom contacts, suggesting interpenetration of the molecular envelopes.

Amino Acid Sequence↗