| dc.description.abstract | The studies described in this chapter have focused on conformational analysis of two antigenic sequences from the N- and C-termini of the chicken riboflavin carrier protein (cRCP). Both sequences investigated have a length of 21 residues and have been conveniently assembled by solid-phase synthesis using Fmoc chemistry. The antibodies raised to both peptides have been shown to cross-react with the native protein cRCP in vitro. Furthermore, antisera raised to both peptides could terminate pregnancy in rodents, establishing their ability to immunoneutralize the protein in vivo.
NMR and CD studies of the N-terminal peptide N-21 (GALEGDTHKANPSPEPNMHEY) have been interpreted in terms of three conformationally well-defined peptide segments corresponding to a short, frayed helical stretch encompassing residues 3–9, a polyproline-like segment around residues 12–14, and another short helical turn at residues 18–20. Most interestingly, the presence of an alternating (X-Pro) sequence repeat stabilizes the polyproline II-like structure. Epitope mapping studies lead to the gratifying conclusion that the antigenic segments map very well to the structured regions of the peptide.
In the case of the C-terminal peptide Y-21, no evidence could be obtained for ordered solution conformations in aqueous media. However, the propensity of the sequence to fold into helical structures is clearly borne out by the dramatic induction of helicity upon addition of 2,2,2-trifluoroethanol. The possibility that a helical conformation was indeed antigenically relevant was further probed by designing and synthesizing four analogs in which helix stabilization was achieved by two independent strategies. One approach involved the introduction of appropriately positioned ion pairs using Lys and Glu residues, while the other utilized the helix-nucleating potential of ?-aminoisobutyric acid (Aib), previously established in this laboratory. All designed analogs show substantial increase in helicity in aqueous-TFE mixtures as compared to the parent peptide. Antisera raised to peptide analogs cross-reacted exceedingly well with the native protein antigen in vitro and were also effective in in vivo immunoneutralization. A most satisfying correlation between helicity as determined by CD and the affinity of the peptide antibodies to the native protein antigen was obtained. The success of the present approach in designing effective synthetic peptide antigens has implications in further development of approaches to immunointerception of pregnancy using riboflavin carrier protein as a reproductive stratagem.
The efficacy of peptide antibodies to N-21 to interfere with RCP function has been shown by both active and passive immunization (Beena, 1994). Preliminary studies with subhuman primates (M. radiata) have also provided evidence that active immunization with the peptide conjugate provides protection from pregnancy establishment over at least five consecutive ovulatory cycles provided the antibody titers are maintained elevated (Adiga et al., unpublished). In terms of the mechanism underlying bioneutralization, it may be pointed out that antibody binding to RCP has no discernable effect on vitamin binding (Murthy et al., 1976). Therefore, it is most likely that the in vivo immunoneutralizing ability arises by blocking crucial sites necessary for receptor recognition on placental membranes (Deviprasad, 1992).
It is intriguing that antisera generated to 21-residue peptides from both amino and carboxy termini (Adiga, 1994) are effective in neutralization of RCP in vivo with consequent pregnancy curtailment in rodents. These results are consistent with the involvement of both the N- and C-terminal segments in receptor recognition. Such a model would require the two termini to be spatially proximate in the three-dimensional structure. Analysis of crystal structures of single-domain globular proteins has shown that the N and C termini are frequently in close proximity (Thornton and Sibanda, 1983).
This thesis advances the design of proteins with tailor-made structures utilizing non-protein amino acids as stereochemical directors of polypeptide folding. The studies presented in this thesis are aimed at evaluating the role of non-protein amino acids like ?-aminoisobutyric acid, C?,?-di-n-propyl glycine, D-residues, etc., in the “Meccano-set” approach to de novo design. An assessment of the conformational preferences of such “directors of polypeptide folding” and their ability to act in tandem to generate defined secondary structures has been made. In the studies presented, the designed peptides revealed the dominating conformational determinant(s) in the final folded structure. The preceding sections of this thesis have described the rationale behind the present approach, the relative merits of this strategy, and the design, synthesis, and conformational characterization of model peptides.
The main results of this thesis are summarized below:
In Chapter 3, a decapeptide (Boc-Aib-Trp-Leu-Aib-Ala-Leu-Aib-Ala-Phe-Aib-OMe, WF10) was designed and synthesized for studying aromatic side chain packing in crystals. The structures of WF10 and its N-terminal nona- (WLF9) and octa- (LF8) peptides yielded a 3_10 ? ?-helical transition in the same octa-peptide segment (Leu-Aib-Ala-Leu-Aib-Ala-Phe-Aib) on going from the eight- to the ten-residue peptide in crystals. Study of these peptides and the N-terminal hepta- (ULF7) and hexa- (LF6) peptides by spectroscopic techniques indicated that infrared spectroscopy has mapped the transition at the level of the octapeptide in the solid state. NMR studies in CDCl3 have, however, yielded evidence supporting a 3_10 helix in all five peptides studied. These studies emphatically show that the two types of helices are nearly isoenergetic, with subtle factors like chain length, Aib content, and environment being responsible for tilting the energy balance between the two types of helices. This study also brings out the limitations of using the NMR method of solvent perturbation of NH chemical shifts in inferring the helix type.
The studies presented in Chapter 4 were undertaken to examine the conformational effect of a tripeptide GDpgX (X = Ala, Leu) when placed at the amino terminus of a previously characterized helical module (Val-Ala-Leu-Aib-Val-Ala-Leu, UV7). At the time when this investigation was initiated, almost all Dpg residues that had been crystallographically characterized in small homopeptides had adopted fully extended (?, ? = 180°) conformations. Spectroscopic studies in solution, together with the crystal structure results, conclusively establish that the Dpg residue has adopted a helical conformation (? ? 60°, ? ? 40°) in both decapeptides. The experimental results suggest that the extended and helical conformations are almost isoenergetic. The greater conformational versatility of the Dpg residues indicates that a complete understanding of precise environmental and sequence effects is necessary before using these residues in the Meccano-set approach towards design of extended polypeptide modules.
A major goal of investigations in this laboratory has been to generate well-characterized helix–helix motifs in synthetic peptides. The use of D-residues and proline in conjunction to act as a helix-breaking segment has been investigated in Chapter 5. Two sixteen-residue peptides are:
Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-D-Phe-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (DFP16)
Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-L-Phe-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (LFP16)
These peptides were designed, synthesized, characterized, and studied in CDCl3 solution by NMR. The strategy aimed at using a D-residue (D-Phe) and a proline residue to interrupt helical folding to generate an ?,?-motif. Detailed analysis of the NOEs in the linking segment showed that these residues had indeed adopted a helical conformation. The presence of Phe(8)NH-C?H2 NOEs clearly points to a helical conformation. Further support for this interpretation comes from delineation of solvent-shielded NH groups, as the Val(10)NH group is strongly solvent-shielded. The results demonstrate that the overwhelming tendency of Aib residues to promote helical folding has prevailed and forced the D-Phe-Pro segment (which has a low propensity to be accommodated in a right-handed helix) into a helical conformation. The ambiguities in inferring helical interruptions from CD data has also been shown. The aromatic contributions from the Phe chromophore to the near-UV CD band had given lower ellipticities to the peptide DFP16 as compared to the control peptide LFP16, suggesting initially that a helical propagation had indeed been interrupted in solution.
Glycine is the most conformationally flexible amino acid. A study of the contrasting conformational preferences of Gly and the strongly helix-promoting Aib was carried out. This study afforded an inspection of the behavior of these residues when present in tandem. The C-terminal heptapeptide fragment of the natural fungal peptide antibiotic Trichogin A-IV (Boc-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe) was investigated for this purpose. 1D NMR studies in CDCl3 using radical line broadening and delineation of solvent-shielded NH chemical shifts showed that Gly(1)NH, Gly(2)NH, Aib(4)NH, and Leu(7)NH were solvent-exposed. Analysis of nuclear Overhauser effects convincingly demonstrated a non-helical multiple ?-turn for this segment. An overlapping Type II/I? was mapped on the Leu-Aib-Gly segment of this peptide in CDCl3. A structural transition to a frayed right-handed helix in (CD3)2SO has also been mapped. The results provide a starting point for the design of overlapping turns. These prompted conformational studies on Trichogin A-IV.
The solution NMR studies on Trichogin A-IV acid (Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OH) revealed that this peptide aggregates in CDCl3. Consequently, the complete conformational analysis on Trichogin A-IV acid was carried out in CDCl3 containing a drop of (CD3)2SO. These studies showed that the peptide was helical under these conditions. The results establish the conformational variability of the Gly-Gly segment.
In Chapter 7, two 21-residue peptides whose sequences had been derived from the N- and C-terminal regions of the chicken riboflavin carrier protein were investigated. The N-terminal peptide N-21 (Gly-Ala-Leu-Glu-Gly-Asp-Thr-His-Lys-Ala-Asn-Pro-Ser-Pro-Glu-Pro-Asn-Met-His-Glu-Tyr) revealed short frayed helical segments in (CD3)2SO as shown by an analysis of nuclear Overhauser effects. Circular dichroism revealed a strong negative band at 204 nm under all conditions investigated. These have been interpreted as originating from polyproline II-like conformation. The conformational assignment from NMR and CD results suggests that three regions of moderately ordered structures may be populated in solution in peptide N-21: a short, frayed helical stretch encompassing residues 3–9, a polyproline-like segment around residues 12–14, and another short helical turn at residues 18–20.
The C-terminal peptide Y-21 (Tyr-His-Ala-Cys-Gln-Lys-Lys-Leu-Leu-Lys-Phe-Glu-Ala-Leu-Gln-Gln-Glu-Glu-Gly-Glu-Glu) showed a propensity to fold in a helical conformation under appropriate conditions. Preceding chapters and earlier studies from this laboratory have conclusively established the helix-nucleating potential of ?-aminoisobutyric acid. These residues were used to impart stereochemical rigidity to this peptide segment of the chicken riboflavin carrier protein. Analogs were subsequently designed to stabilize the helical conformation by either the introduction of appropriately positioned salt bridges or the helix-nucleating potential of ?-aminoisobutyric acid. The results showed all designed analogs (Y-21, EC-21, HE-21, HC-21) are more helical than the parent peptide. A clear correlation between structure and the affinity of the peptide antibodies to the native protein antigen has been obtained.
Engineering of antigenic determinants on supersecondary structures using de novo design approaches often involves synthesis of long peptide chains (35–80 residues long). This chapter illustrates that stabilization of secondary structure by rational design can also greatly enhance immunogenicity and antigenicity but in much shorter peptide sequences (21 residues long). The success of the present approach in designing effective synthetic peptide antigens has implications in development of approaches to immunointerception of pregnancy using riboflavin carrier protein as a reproductive stratagem.
The studies described in this thesis underline the need to further characterize the conformational preferences of structure-directing residues when they are present in tandem. The use of these non-protein residues for nucleating polypeptide folds (Figure 8.1) has been emphatically illustrated in the stabilization of a helical conformation of the C-terminal 21-residue peptide of the chicken riboflavin carrier protein. These studies permit an evaluation (albeit preliminary) of the utility of these residues in the design and construction of synthetic protein mimics. Clearly, considerable effort is necessary before the stereochemical preferences of these non-protein residues can be exploited in the design of synthetic proteins. | |
