|
Protease-catalyzed
splicing of peptide bond |
| Principal Investigator : Rajendra P Roy
Project
Associates/Assistants
Ph
D Students The
theme of research is to study the underlying principles and mechanisms of
reverse proteolysis, and also apply protease-mediated peptide ligation
reactions for semi-synthesis of proteins. Currently, this strategy is being
used to engineer new and novel hemoglobin molecules to delineate
intermolecular interactions in the assembly of the sickle hemoglobin fiber. A.
Protease-catalyzed splicing of peptide fragments The
objective of the study is to carry out model studies on protease-mediated
peptide ligation and protein semisynthesis. Although
RNase A could not be formed from its complementary fragments [S-peptide
(residues 1-20) and S-protein (residues 21-124)] in the crowded milieu,
subtilisin-mediated splicing of 20-21 peptide bond readily occurs in the
presence of organic cosolvent such as glycerol. We have used this strategy to
introduce fluoroPhe residues in RNase A with the idea that studies of
semisynthetic RNase A may serve as a model system and provide a general sense
of the effect of fluoroPhe residues on structure and function of proteins. The
role of Phe at the 8th
position towards stability and catalysis of RNase A was investigated through
the introduction of isosteric fluorophenylalanine residues. The Phe8 residue
is in sequence contiguity with Lys7 that together with Arg10 forms the P2
substrate subsite. His12, one of the catalytic residues in RNase A is also in
close proximity (~ 4Å) to Phe8. We wished to explore if interactions due to
Phe8 could be perturbed by alterations of the charge density of the aromatic
ring. Two analogs of phenylalanine; 3-fluoro-L-phenylalanine and
3,4-difluoro-L-phenylalanine, were used so that a gradation in the effects of
hydrophobic and electronic changes can be brought about in the region of RNase
A that is sensitive to substrate binding and catalysis. The three synthetic
peptides, native S-peptide, SFx (S-peptide with 3-fluoro-L-phenylalanine at
the 8th
position) and SFxx (S-peptide with 3,4-difluoro-L-phenylalanine at the 8th
position), respectively, were ligated with S-protein to generate native RNase
A and the mutant proteins (SFx-RNase A and SFxx-RNase A). The proteins were
obtained in pure form. The
ability of fluoroPhe residues to impact the polypeptide conformation was
assessed in the S-peptide and RNase A. S-peptide and its analogs, although,
almost structure-less in aqueous solution, exhibited significant helical
structure in the presence of structure-promoting organic co-solvent. The CD
spectra monofluoro derivative was identical to that of S-peptide suggesting
that the introduction of fluorine atom in the phenyl ring did not affect the
stability of the peptide. However, a slight decrease in the helicity was
noticeable in the difluoro derivative indicating that the two fluorine atoms
substituted in the adjacent positions of the Phe8 may have perturbed the
peptide stability by de-stabilizing Phe8-His12 interactions. Although, this is
in consonance with the fact that His12+
and Phe8 interaction in the C-peptide stabilizes the a-helix,
the effects observed in the mutant peptides are rather modest. Interestingly,
this behavior was maintained even at the level of protein in that the thermal
melting profiles of mutant proteins (SFx-RNase A and SFxx-RNase A) were almost
identical to the wild type enzyme. Thus, introduction of fluoroPhe residues do
not seem to affect the conformational stability of RNase A in any significant
fashion. Studies on the effect of fluoroPhe residues on enzyme kinetics are
currently in progress. B.
Chemo-enzymic engineering of sickle hemoglobin The
objective of the project is to studysite-specific incorporation of natural
and/or nonstandard amino acids in hemoglobin to probe the intermolecular
interactions in sickle hemoglobin (HbS) fiber. During
the reporting period, studies were focused to delineate the inhibitory
mechanism of Gly mutants. Although, sites 19 and 21 are not involved in any
fiber contact but their replacements with Gly residues generate a sequence
(residues 18-22, G-G-H-G-G) that has the propensity to enhance the
conformational flexibility of the AB region. The AB region of a-chain
contains several contact sites of the fiber (a16,
a20
and a23)
and lies in spatial proximity to a cluster of GH corner residues that are
implicated in fiber interactions (residues a113
through a116).
Besides, AB-GH corners of the a-chain
engage in extensive inter-tetrameric interactions with AB-GH corners of the b-chain
yielding physiologically important intra-double strand axial contacts of the
fiber. Therefore, it is conceivable that even subtle alteration of the
conformational dynamics generated through rational amino acid changes at non-contact
sites at the AB or GH corners of a/b
chains might influence the HbS polymerization by perturbing the inter-tetrameric
interaction interface of the fiber. Toward this, we have carried out dynamics
investigations of the Gly mutant chain. Additionally, we have engineered a-aminoisobutyric
acid (Aib) residues at 19 and 21 positions to compare and contrast the
influence of Gly residues at the same site. MD
simulations of mutant a-chains
show that both, Gly and Aib mutants, perturbed the dynamics of the AB and GH
region of the chain. However, distribution of Ca-Ca
distances between pairs of AB-GH residues indicated that Gly mutant exhibit
greater fluctuations as compared with Aib and Ala (native chain). The
distribution of Ca-Ca
distance between Ala13 and Glu116 is more variable in the Gly mutant and tends
towards larger values. A similar trend is also apparent in the Ca-Ca
distance distribution calculated for the Ala13-Leu113 pair. In the case of the
Lys16-Glu116 and Lys16-Leu113 pairs, the effect is less pronounced for the Ca-Ca
distances, but is clearly visible when one considers the side chain atoms NZ
of Lys16 and CD of Glu116. The dynamical cross-correlation maps (DCCM) of
native (Ala) and Aib substituted a-chains
showed strong motional coupling of AB and GH region residues, whereas that of
Gly substituted chains displayed significantly less coordinated movement. This
can be interpreted to mean that AB-GH region of native or Aib mutant chains
may be more rigid than the Gly mutant chain. However, Aib residue may exert
similar effects as that of the Ala. Interestingly, the polymerization behavior
of Aib mutant was found to be very similar to native HbS. Previously
we had reported the polymerization behavior of the aforementioned triple and
double mutants and deduced that the interactions emanating from mutations of
the AB and EF regions must be additive. We determined the polymer solubility (Csat)
of HbS containing only the point mutation of a Gln or His for Asn at a78
to assess the individual contribution of these residues in the polymerization
of HbS. The Csat
of a2(Q78)b2S
mutant was found to be similar to that of the native HbS suggesting that Gln
residue behaves in much the same way as Asn. In contrast, the HbS mutant [a2(H78)b2S]
containing His at this site yielded a Csat
that was significantly lower than that of native HbS. This value of Csat
was far less than that expected from an additive contribution of double [a2(G19G21)b2S]
and single mutant [a2(H78)b2S]
in the triple mutant. The shift in Csat
of the triple mutant clearly suggested that the polymerization-enhancing
effects due to the presence of His-78 at the EF corner synergistically
compensated for the inhibitory effect, emanating from the Gly substitutions at
the AB corner. Interestingly, His-78 shifted the Csat
of double mutant [a2(Q16H78)b2S]
in a similar synergistic fashion as it did in the case of the triple mutant [a2(G19G21H78)b2S].
Thus the polymer solubility measurements of the point mutant HbS (His-78)
unambiguously established the cooperative linkage of AB and EF region fiber
interactions. In
summary, the ability of a polymerization-enhancing mutation of an established
inter-double strand contact point (aHis-78)
at the EF corner of the a-chain
to completely neutralize the inhibitory effect of Gly substitutions at the AB
corner is an interesting result that suggest long-range and non-additive
coupling of mutational effects in HbS polymerization. This view is
strengthened with the observation that the coupling of aHis-78
at the EF corner to mutation of a specific intra-double strand fiber contact (a16)
in the AB region occurs in a similar synergistic fashion as it happened with
Gly mutants. The fact that aLys-16
makes axial contacts with the GH corner residues (a114,
a115),
and Gly substitutions at the AB corner exert their influence on polymerization
by inducing dynamic changes in the AB-GH region of the a-chain,
raises the intriguing possibility of interaction-linkage between AB, GH and EF
regions of the a-chain
in the HbS fiber assembly. Our previous work demonstrating that the inhibitory
effect of a16
was additive with a113
(GH1) is consistent with this idea.
Publications
Original
peer-reviewed articles 1.
Sudha R, Anantharaman L,
Sivaram MV, Mirsamadi N, Choudhury D,
Lohiya NK, Gupta RB and Roy RP (2004) Linkage of interactions in sickle
hemoglobin fiber assembly: Inhibitory effect emanating from mutations in the
AB region of the a-chain
is annulled by a mutation at its EF corner. J Biol Chem (in press).
Reviews
/Proceedings 1. Balajee SR, Kumaran S, Datta D and Roy RP (2004) Protein splicing by reverse proteolysis. In: Deep roots, open skies: New biology in India. (Eds. Basu SK, Batra JK and Salunke DM) Narosa Publishing House, New Delhi, 141-145. |