Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

Abstract

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.

Andrographolide, a Novel NF-κB Inhibitor, Induces Vascular Smooth Muscle Cell Apoptosis via a Ceramide-p47phox-ROS Signaling Cascade

“…Recent studies have demonstrated that ceramide increased in endothelial cells exposed to death factors, including tumor necrosis factor α , interleukin 2, and endostatin, and in ischemic reperfused myocardium [46, 47]. In these studies, the ceramide signaling pathway has been confirmed to be involved in the activation of Nox and consequent O 2 − production [48, 49]. Ceramide also enhances ROS formation in mammalian cells by straightly raising the permeability of mitochondrial membranes to cytochrome c [50] and restraining the isolated mitochondria complex III [51].…”

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

“…They are also extensively used for studying enzyme active centres (Ma et al, 2002;Seeley et al, 2005) and as catalysts of enzymatic synthesis (Bonger and Lambros, 1994;Wehofsky et al, 2001). For instance, proteinase V8 from Staphylococcus aureus is capable of catalysing the oligomerization reaction of native protein segments of ribonuclease, as well as condensation of complementary fragments of human haemoglobin molecule (Sahni et al, 1998;Kumaran et al, 2000). Glutamyl endopeptidases can also be utilised for the synthesis of synthetic peptides used as substrates for proteinases (Braddam and Meldal, 1992).…”

Improvement of Staphylococcus aureus‐V8‐protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides

“…However, the obtention of these two peptides from haemoglobin has never been reported. V8 protease has been widely employed for fragmentation of proteins prior to amino‐acid‐sequence determination and as catalyst for the synthesis of specific peptide bonds [13–16]. It has proved to be eminently suitable for generating digests which contain many appropriately sized peptide fragments, as it exhibits a high degree of specificity for the Glu and Asp residues of peptides and proteins [17,18].…”