Abstract
The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the α-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated α-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.
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Abbreviations
- Hb:
-
hemoglobin
- HbS:
-
sickle hemoglobin
- HMB:
-
hydroxymercuricbenzoic acid
- FPLC:
-
fast protein liquid chromatography
- RPHPLC:
-
reverse-phase high performance liquid chromatography
- MD:
-
molecular dynamics
- CD:
-
circular dichroism
- Far-UV:
-
far-ultraviolet
- DFD:
-
distance frequency distribution
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Acknowledgements
The first two authors (SB and NM) have contributed equally to this work. SB and LA are Senior Research Fellows of the Council of Scientific and Industrial Research, India. NM is a recipient of a fellowship from the University Grants Commission (UGC), India. This work was supported by core grants to the NII from the Department of Biotechnology, Government of India, and to JNU by the UGC under its UPOE program.
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Banerjee, S., Mirsamadi, N., Anantharaman, L. et al. Modification of Axial Fiber Contact Residues Impact Sickle Hemoglobin Polymerization by Perturbing a Network of Coupled Interactions. Protein J 26, 445–455 (2007). https://doi.org/10.1007/s10930-007-9084-1
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DOI: https://doi.org/10.1007/s10930-007-9084-1