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Protease-catalyzed splicing of peptide bond |
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Principal Investigator : Rajendra P Roy Ph D students The
two major themes of the project are i) understanding the structural and
mechanistic imperatives of peptide ligation reactions catalyzed by proteases
and ii) chemo-enzymic engineering of new and novel hemoglobin tetramers to
delineate the intermolecular interactions in the sickle hemoglobin fiber. A. Protease-catalyzed splicing of native peptide fragments Model studies of reverse proteolytic condensation reactions utilizing
natural or designer peptide segments were continued. During the current year,
we investigated the influence of macromolecular crowding on the
protease-catalyzed peptide bond equilibrium. The rationale, that volume
exclusion by an inert background macromolecule admixed with a protein would
shift the equilibrium towards attaining a smallest excluded volume and favour
formation of compact structures, formed the basis of the studies. We surmised
that protease-mediated synthesis of coiled-coil or helix bundle type of
structures from the respective constituent peptide fragments should be
facilitated in the presence of crowded dextran solution as intermolecular
interactions leading to association of enzymically ligated molecules into such
ordered structure would result in a reduction of excluded volume. Three 20 residues designer peptides, each containing Glu at the
eleventh position to ensure specificity of V8 protease, were employed for this
study; two of these peptides were shown to exhibit coiled-coil structures
while the third one was a random coil control. The V8 protease-catalyzed
ligation of respective complementary fragments (residues 1-11 and 12-20) of
both the coiled-coil peptides proceeded smoothly, albeit with different
extents, in the presence of dextran. In contrast, under similar conditions,
the complementary fragments of the random coil peptide could not be ligated.
These results suggest that enhanced tendency of polypeptide chains to form
compact complexes in crowded media could be exploited to facilitate reversal
of proteolysis. B. Chemo-enzymic engineering of proteins We had constructed mutants of hemoglobin with a view to probe the
intermolecular interactions in sickle hemoglobin (HbS) fiber. Detailed studies
of HbS (a113His
®Leu)
construct was carried out to unequivocally establish a role for a113
amino acid residue in the polymerization process. For the sake of brevity,
henceforth the above mutant HbS would be referred to as HbS Twin Peaks after a
natural hemoglobin variant. The global conformation of HbS Twin Peaks with
respect to the environment of heme, as judged by soret region CD spectra, was
found to be similar to that of the native HbS. First derivative UV spectra for
liganded and unliganded forms of HbS Twin Peaks, which serve as indicators of
micro-environment of aromatic residues located at the a1b2
interface (Trp37b
and/or Tyr42a),
were also similar to HbS indicating that HbS Twin Peaks assumes HbS-like
quaternary structure. The preservation of quaternary structure and dimer-interaction
in HbS Twin Peaks was further corroborated by oxygen binding measurements.
Both the P50
(oxygen affinity) and Hill coefficient (extent of co-operativity) of HbS Twin
Peaks were similar to HbS. The kinetics of polymerization revealed that the
Twin Peaks mutation impeded the rate of gelation of HbS. This result was
consistent with the polymer solubility (Csat)
data; Csat
of HbS Twin Peaks was found to be about 1.8 fold higher than that of native
HbS. In order to further authenticate the participation of a113
site in the polymerization process as well as to evaluate its relative
inhibitory strength, we constructed HbS tetramers in which a113
mutation at the GH corner was coupled individually with two established fiber
contact sites (a16
and a23)
located in the AB region of the a-chain;
HbS (a16Lys®Gln,
a113Leu®His),
HbS (a23Glu®Gln,
a113Leu®His).
The single mutants at a16/a23
sites were also engineered as controls. The Csat
values of the HbS point mutants involving sites a16
or a23
were higher than HbS but markedly lower as compared with HbS Twin Peaks. In
contrast, Csat
of both the double mutants were comparable to or higher than HbS Twin Peaks
indicating independent or additive inhibitory effect. The demonstration of
inhibitory effect of a113
mutation alone or in combination with other sites, in quantitative terms,
unequivocally establishes a role for this site in HbS gelation. a113
amino acid residue is not postulated in any interactions in the crystal
structure of HbS and is also excluded by the models of the HbS fiber. Further
solution co-polymerization studies of HbS Twin Peaks should help delineate the
stereochemistry of a113
residue in the polymer and facilitate efforts to develop an accurate model of
the HbS fiber that could serve as a blueprint for therapeutic intervention. 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 inhibitory
strength and interaction-linkage with two fiber contact sites (a16/23) located in the AB region of the
a-chain. J Biol Chem (in press). 2.
*Kumaran S, Datta D and Roy RP (2000) An enigmatic peptide ligation reaction:
Protease-catalyzed oligomerization of a native protein segment in neat aqueous
solution. Protein Science 9:734-741. (*in press last year, since published). |