Influence of semisynthetic modification of the scaffold of a contact domain of HbS on polymerization: role of flexible surface topology in polymerization inhibition.

A new variant of HbS, HbS-Einstein with a deletion of segment α in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain.

HbS-Savaria: the anti-polymerization effect of a single mutation in human alpha-chains.

Recombinant alpha-Savaria globin (alpha(S49R)) was assembled with beta(S) chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (alpha (2) (S49R) beta (2) (E6V) ) that exhibited normal O(2) affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O(2) affinity by 15%.

Pair-wise interactions of polymerization inhibitory contact site mutations of hemoglobin-S.

The linkage of pair-wise interactions of contact site mutations of HbS has been studied using Le Lamentin [His-20 (alpha)-->Gln], Hoshida [Glu-43 (beta)-->Gln] and alpha(2)beta (2) (T87Q) mutations as the prototype of three distinct classes of contact sites of deoxy HbS fiber. Binary mixture experiments established that beta(A)-chain with the Thr-87 (beta)-->Gln mutation is as potent as the gamma-chain of HbF (alpha(2)gamma(2)) in inhibiting polymerization. On combining the influence of Le Lamentin mutation with that of beta (2) (T87Q) mutations; the net influence is only partial additivity.

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain.

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect.

Product conformation driven splicing of unprotected peptides by reverse proteolysis: influence of intrinsic and extrinsic factors.

The structural motif of 'product conformation driven V8 protease catalyzed ligation reaction' can be represented by FR(I)-EALER-FR(II). The relative roles of the flanking regions (FR(I) and FR(II)) and of splicedon, the central penta-peptide, on the thermodynamic stability of the 'conformational trap' of the product has been now evaluated as a function of co-solvent concentration. The studies have established that the thermodynamic stability of the conformational trap of alpha17-40des23-26 with four different splicedons (EALER, EALEV, EYGER, or EGAER) that differ in the intrinsic alpha-helical potential of their amino acid residues and/or ability to generate i, i+4 side chain interaction is a direct correlate of the n-propanol induced alpha-helical conformation of the product.

Product-conformation-driven ligation of peptides by V8 protease.

Organic co-solvent-induced secondary conformation of alpha(17-40) of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of alpha(17-40) has been conceptualized by the general structure FR(I)-EALER-FR(II) and the pentapeptide sequence EALER playing a major role in inducing the alpha-helical conformation. The primary structure of alpha(17-40) has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated.