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Protease-catalyzed splicing of peptide bond |
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Principal Investigator : Rajendra P Roy Research
Associate
PhD
students
The
two major themes of the project are A) understanding the structural and
mechanistic imperatives of peptide ligation reactions catalyzed by proteases
and, B) engineering of new and novel hemoglobin tetramers to delineate
intermolecular interactions in the sickle hemoglobin fiber. A.
Protease-catalyzed splicing of native peptide fragments Model
studies on reverse proteolytic condensation reactions utilizing natural or
designer peptide segments is the objective of the research. In
the reporting year, we explored if the volume exclusion effect or the ability
of the crowding molecules to strengthen the associative interactions between
non-covalently held segments could facilitate the splicing of discontiguity
sites in fragment complementing systems. RNase A and triosephosphate isomerase
(TIM) were chosen as model system for this study. The respective proteins were
proteolyzed by subtilisin in dilute aqueous solution and their re-formation
followed in the presence of crowded media such as polyethyleneglycol (PEG) and
dextran. The ligation reactions were also carried out in the presence of
organic cosolvents to serve as controls. The
multiply nicked (six or more fragments) TIM was converted into intact TIM in
the presence of dextran or PEG albeit with slower kinetics as compared
with that observed in the presence of aqueous-acetonitrile solution.
Interestingly, a single nick in RNase A could not be ligated under similar
conditions in the crowded milieu. The control reaction in the presence of
glycerol proceeded smoothly. The failure of synthesis in the case of RNase A
suggest that conversion of RNase S to RNase A may not be accompanied by
sufficient reduction in volume exclusion. This is consistent with the fact
that the crystal structures of RNase S and RNase A are almost superimposable.
In contrast, considerable compaction, and consequently large volume reduction
is attained in the case of TIM re-formation. B.
Chemo-enzymic engineering of proteins Site-specific
incorporation of natural and/or nonstandard amino acids in hemoglobin to probe
the intermolecular interactions in sickle hemoglobin (HbS) fiber is the
objective of this work. In
the reporting year, further studies on the polymerization of HbS twin peaks [HbS
(a113His®Leu)] were done with the idea to
delineate the quinary interactions of a113 site in the HbS
fiber. Towards this, we constructed HbA Twin Peaks and carried out
co-polymerization studies. The rationale of the study was based on the fact
that while a deoxygenated stoichiometric mix of HbA twin peaks and HbS would
be largely populated by tetramers with a113 site in the trans
configuration, a similar mixture containing HbS twin peaks and HbA would be
dominated by tetramers with the mutant site in the cis orientation.
Thus a comparative analysis of equilibrium solubility (Csat) of these mixtures
with that of the HbS/HbA mixture would provide insights into the relative
contributions of the cis/trans stereochemistry of the a113
site in the polymerization of HbS. The Csat value of the ‘trans’ mixture
was found to be similar to that of the control HbA/HbS mixture suggesting that
the site was inactive in the trans configuration. Consistent with this,
the ‘cis’ mixture yielded a Csat that was significantly higher than the
control sample. These results establish that a113 site facilitates
the fiber assembly only when present in the cis orientation i.e
in the same ab dimer that contains the ‘active’ val6b. We
earlier reported the studies on the interactions between a113
with a16 or a23 amino acid residues, proximal sites
located in the AB-GH region of the molecule. We observed that the inhibitory
effect of a113 was additive with a16
but not with a23. In our continued efforts to
delineate interaction-linkage map of participating residues, we initiated
studies to examine concerted interactions, if any, between two well separated a-chain
contact sites. Two sites, a16 (AB) and a78
(EF) were chosen for this study. We intended to use the chemo-enzymatic
strategy for the construction of a-globin mutants,
namely, the propensity of V8 protease to catalyze the ligation of
complementary fragments, a1-30 and a31-141 to generate a full-length a-globin
(a1-141). We wished to introduce mutation at a16
through a synthetic a1-30 segment and that at a78
by using the a31-141 fragment from the a-chain
of Hanuman Langur (Presbytis entellus) hemoglobin. The reported
sequence of the a-chain of langur differs from human at only three sites; a19Ala®Gly,
a21Ala®Gly, and a78Asn®His. The
Hb samples from Langur, surprisingly, yielded two chromatographically distinct
a-chains.
The mass spectrometric analysis, tryptic peptide mapping and amino acid
sequencing of the tryptic peptides lead to the identification of a variant
that differed from the documented sequence only at a78
site (His®Gln). The discovery of this new variant provided us with an
opportunity to probe the influence of the microenvironment of this site on the
polymerization reaction. Accodingly, we prepared respective hybrid tetramers
of the two chains with the bs-chain.
Both the HbS hybrid tetramers (a2
78Asn®His b2S, and a2
78Asn®Gln b2S) displayed near UV and soret region CD spectra
that was very similar to native HbS. The oxygen binding properties as
reflected by the P50 (half-maximal binding)
value was found to be normal. However, preliminary results of polymerization
studies appear to reveal differences in the behavior of the two hybrids.
Detailed studies on kinetics of polymerization and equilibrium solubility of
the hybrids and the preparation of the double (a16,
a78) mutant HbS are in progress. Publications Original
peer-reviewed articles 1. *Sivaram MVS, Sudha R and Roy RP (2001) A role for a113 (GH1) amino acid residue in the polymerization of sickle hemoglobin: Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (a16/23) in the AB region of the a-chain. J Biol Chem 276:18209-18215 (*in press last year, since published). |