Intersubunit circular permutation of human hemoglobin

Abstract

For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species. The use of total gene synthesis has resulted in very high levels of functional human Hb expression in Escherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight. In this communication, we report a novel method for linking alpha chains. By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity. The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic. IntroductionA mechanistic and predictive understanding of how a primary sequence can fold to a single tertiary structure remains undefined. Since a thorough understanding of this process will enable the rational design of unique protein structures, this remains an area of active research. One theory suggests that short continuous regions within a protein develop local interactions early in the folding process to minimize the number of accessible structures and catalyze the formation of a functional protein. Radical perturbations of protein structure by circular permutation is one technique being used in an attempt to explore this issue. In addition, utilization of circular permutation itself can be exploited in the design of new protein structures.A circularly permuted protein is created in 2 steps. First, the original termini are linked to form a circular polypeptide. New termini are then created by cleavage of a peptide bond at a location distant from the original termini. Goldenberg and Creighton engineered the first circularly permuted sequence by chemically condensing the termini of bovine pancreatic trypsin inhibitor (BPTI) and generating new termini by limited proteolysis. 1 This circularly permuted variant was shown to refold, in vitro, to a native functional conformation. This seminal experiment suggested that the location of the termini has little effect on the final 3-dimensional structure of the protein. Luger and coworkers genetically circularly permuted phosphoribosyl anthranilate isomerase. 2 The Escherichia coli translated variant was structurally and functionally similar to the wild-type protein. Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic cha...

The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane

“…Membrane-bound hemoglobin was measured using the pyridine hemochromagen assay of Sanders et al [38] . To quantify the spectrin present, ghosts were prepared from washed erythrocytes as described above and then treated with solubilisation buffer (125 mM Tris-HCl pH 6.8; 4% SDS; 20% glycerol; 10% 2-mercaptoethanol) and heat denatured at 95 °C for 5 min.…”

Coexpression of Human α- and Circularly Permuted β-Globins Yields a Hemoglobin with Normal R State but Modified T State Properties

“…Precedent for this idea comes from the work of Sligar and co-workers, who reported the circular permutation of a di-α-globin sequence to yield a functional Hb (36). Our laboratory has shown previously that relocating the termini in swMb to the G-H loop by circular permutation does not appear to significantly alter the structure of the heme binding pocket or its ligand binding properties (37).…”

“…Our results show that ligand binding to the α-cp β dimer exhibits almost identical properties (Figure 6). Sanders et al (36) reported that a recombinant hemoglobin derived from a circularly permuted di-α-chain associated with two wild-type β -globin subunits has an increased O 2 affinity ( P 50 = 0.9 mmHg) and decreased cooperativity ( n = 2.0) compared to HbA. The higher cooperativity of this permuted di - α - β 2 hemoglobin, compared to α-cp β is likely achieved as a result of stable quaternary contacts between the four globin subunits, which are not present in a dimer.…”

“…Hemoglobins with covalently linked di-α-chains (10), circularly permuted di-α-chains (36), and covalently linked di-α- and di- β -chains (35) have been described. However, a single Hb gene containing two α and two β domains has not been investigated as a possible HBOC.…”

Conditioning Regimens Including High-Dose Busulfan Cause a High Incidence of Transplant-Related Mortality after Myeloablative Stem Cell Transplantation

“…These alternatives to BU treatment may solve problems and become breakthroughs for stem cell transplantation in patients with advanced disease. Indeed, recent reports show favorable allogeneic transplantation outcomes using intravenous or targeted oral BU conditioning regimens [23, 24, 25]. …”