Expression of Recombinant Monomer Hemoglobins (Component IV) from the Marine Annelid Glycera dibranchiata: Evidence for Primary Sequence Positional Regulation of Heme Rotational Disorder

Abstract

A description of the efficient high-level expression of the monomer hemoglobin (GMG4) from Glycera dibranchiata is presented. The cDNA described by Simons and Satterlee [Simons, P.C., & Satterlee, J.D. (1989) Biochemistry 28, 8525-8530] was subcloned into an expression system, and conditions were found that led to the production of large amounts of soluble apoprotein (rec-gmg). These conditions included lowering the temperature during the induction period and growth in a rich medium with a higher ionic strength. Characterization of this reconstituted recombinant protein showed that it was not identical to the native GMH4 protein. Both UV-visible and 1H NMR data indicated differences within the holoprotein (rec-gmh) heme pocket compared to the native protein, the major difference being that two nonidentical heme orientations are significantly populated in rec-gmh. This phenomenon has been seen previously in other heme proteins, where these heme orientational isomers are described by a 180-deg rotation about the heme alpha-gamma meso axis. This work prompted the production of a complete chemical sequence for the native GMH4 [Alam S.L., Satterlee, J. D., & Edmonds, C. G. (1994) J. Protein Chem. 13, 151-164], which showed that the expressed rec-gmg protein differed at three primary sequence positions (41, 95, and 123) from the native component IV globin (GMG4). Subsequently, we have produced the triple-revertant mutations required to express the recombinant wild-type protein (recGMG4). The physical characteristics of the active site in the holoprotein (recGMH4) are identical to those of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cushioning the pressure vibration of a zeolite concentrator system using a decoupled balancing duct system

“…A clear picture of the extent of globin heterogeneity in this organism has emerged only recently, as a result of primary sequencing5, 6 and mass spectrometry 5–7. Those results have confirmed the individual uniqueness and homogeneity of the three major monomer hemoglobins, called components II, III, and IV (or alternatively GMH2, 3, and 4) that have been reproducibly isolated and characterized in this laboratory 3–8, 11–14, 24–39. Recently, the solution structure of the CO‐ligated Fe 2+ (reduced, ferrous) form of recombinant GMH4 (GMH4CO) was determined,38, 39 making it the first structure to be determined of one of our highly characterized monomer hemoglobins.…”

“…Whereas the functional implications of this substitution were immediately clear, the true extent of the globin heterogeneity present in these cells was unrecognized 15–23. Vinogradov and co‐workers1 were the first to separate the G. dibranchiata erythrocyte contents into monomeric and polymeric hemoglobins, but further realization of extensive heterogeneity in the monomeric globin fraction was not recognized until the work of Parkhurst et al2 and our group 3–8, 11–14…”

“…The coelomic erythrocytes of the marine annelid Glycera dibranchiata , commonly named the “blood worm,” contain a mixture of monomeric and polymeric hemoglobins 1–14. Interest in these proteins originated in 1972 when the first primary sequence was published and indicated, by sequence alignment, an exceptional amino acid substitution of His E7† by Leu 15.…”

“…These are naturally isolated not recombinant proteins. Whereas the ferric forms are not physiologically functional, the rationale for determining these structures is that the heme paramagnetism of both unligated and CN‐ligated GMH proteins has been most useful in previous extensive NMR studies3, 4, 13, 14, 28, 29, 33, 35–37 and that cyanide binding kinetics have been measured for these proteins 31, 32. Structural and dynamics studies of native and recombinant GMH proteins are continuing, hence these structures will provide an important standard by which to compare and interpret future results.…”

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor

“…18 The studies demonstrated that K A/B is determined by steric interactions of the peripheral side chains attached to pyrrole rings I and II of the heme, i.e., the side chains at positions 2, 3, 7, and 8 (Figure 1), with the surrounding amino acid residues in the heme pocket. 11,14,18,29,30,36,41 In fact, K A/B has been shown to be greatly affected by replacement of the amino acid residues in close proximity to these heme side chains. 14,18,41 The heme orientational disorder demonstrated that the molecular recognition upon insertion of the heme into the Mb heme pocket is not as strict as that for the formation of usual enzyme−substrate complexes.…”