Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Design of a Peptide-Based Model Leads for Scavenging Anions.

Among several peptide-based anion recognition motifs, the "CαNN" motif containing C-1 α, N0, and N+1 of three consecutive residues is unique in its mode of interaction. Having a spatial geometry of ßαα or ßαß, this motif occurs in the N terminus of a helix and often found at the functional interface of a protein, mediating crucial biological significance upon interaction with anion(s). The interaction of anion(s) with chimeric peptide sequences containing the naturally occurring "CαNN" motif (CPS224Ac, CPS226, and CPS228) reported in our previous attempts strongly confirms that the information regarding the interaction is embedded within the local sequences of the motif segment. At these prevailing circumstances, an effort has been pursued to design novel scaffolds based on the "CαNN" motif for achieving better recognition of anion(s). Exploring the existing data set of the "CαNN" motif available in the FSSP database, four novel peptide-based scaffolds have been designed (DS1, DS2, DS3, and DS4), and preliminary screenings have been performed using computational approaches. Our initial work suggests that two (DS1 and DS3) out of the four scaffolds are potential candidates for better anion recognition. By employing biophysical characterization using both qualitative and quantitative measures, in this present study, we report the interaction of sulfate and phosphate ions with these two designed scaffolds, in which there is much better recognition of anions by these scaffolds than the natural sequences, justifying their logical engineering. Our observation strongly suggests that these designed scaffolds are better potential candidates than those of the naturally occurring "CαNN" motif in terms of anion recognition and could be utilized for the scavenging of anion(s) for different purposes.

Interaction landscape of a 'CαNN' motif with arsenate and arsenite: a potential peptide-based scavenger of arsenic.

Arsenic (As) is a toxic metalloid that has drawn immense attention from the scientific community recently due to its fatal effects through its unwanted occurrence in ground water around the globe. The presence of an excess amount of water soluble arsenate and/or arsenite salt (permissible limit 10 µg L-1 as recommended by the WHO) in water has been correlated with several human diseases. Although arsenate (HAsO4 2-) is a molecular analogue of phosphate (HPO4 2-), phosphate is indispensable for life, while arsenic and its salts are toxic. Therefore, it is worthwhile to focus on the removal of arsenic from water. Towards this end, the design of peptide-based scaffolds for the recognition of arsenate and arsenite would add a new dimension. Utilizing the stereochemical similarity between arsenate (HAsO4 2-) and phosphate (HPO4 2-), we successfully investigated the recognition of arsenate and arsenite with a naturally occurring novel phosphate binding 'CαNN' motif and its related designed analogues. Using computational as well as biophysical approaches, for the first time, we report here that a designed peptide-based scaffold based on the 'CαNN' motif can recognize anions of arsenic in a thermodynamically favorable manner in a context-free system. This peptide-based arsenic binding agent has the potential for future development as a scavenger of arsenic anions to obtain arsenic free water.

Ratio of ellipticities between 192 and 208 nm (R1 ): An effective electronic circular dichroism parameter for characterization of the helical components of proteins and peptides.

Circular dichroism (CD) spectroscopy represents an important tool for characterization of the peptide and protein secondary structures that mainly arise from the conformational disposition of the peptide backbone in solution. In 1991 Manning and Woody proposed that, in addition to the signal intensity, the ratio between [θ]nπ* and [θ]ππ*ǁ ((R2 ) ≅ [θ]222 /[θ]208 ), along with [θ]ππ*⊥ and [θ]ππ*ǁ ((R1 ) ≅ [θ]192 /[θ]208 ), may be utilized towards identifying the peptide/protein conformation (especially 310 - and α-helices). However, till date the use of the ratiometric ellipticity component for helical structure analysis of peptides and proteins has not been reported. We studied a series of temperature dependent CD spectra of a thermally stable, model helical peptide and its related analogs in water as a function of added 2,2,2-trifluoroethanol (TFE) in order to explore their landscape of helicity. For the first time, we have experimentally shown here that the R1 parameter can characterize better the individual helices, while the other parameter R2 and the signal intensity do not always converge. We emphasize the use of the R1 ratio of ellipticities for helical characterization because of the common origin of these two bands (exciton splitting of the amide π→ π* transition in a helical polypeptide). This approach may become worthwhile and timely with the increasing accessibility of CD synchrotron sources.

Computational design of model scaffold for anion recognition based on the 'Cα NN' motif.

The 'novel phosphate binding 'Cα NN' motif', consisting of three consecutive amino acid residues, usually occurs in the protein loop regions preceding a helix. Recent computational and complementary biophysical experiments on a series of chimeric peptides containing the naturally occurring 'Cα NN' motif at the N-terminus of a designed helix establishes that the motif segment recognizes the anion (sulfate and phosphate ions) through local interaction along with extension of the helical conformation which is thermodynamically favored even in a context-free, nonproteinaceous isolated system. However, the strength of the interaction depends on the amino acid sequence/conformation of the motif. Such a locally-mediated recognition of anions validates its intrinsic affinity towards anions and confirms that the affinity for recognition of anions is embedded within the 'local sequence' of the motif. Based on the knowledge gathered on the sequence/structural aspects of the naturally occurring 'Cα NN' segment, which provides the guideline for rationally engineering model scaffolds, we have modeled a series of templates and investigated their interactions with anions using computational approach. Two of these designed scaffolds show more efficient anion recognition than those of the naturally occurring 'Cα NN' motif which have been studied. This may provide an avenue in designing better anion receptors suitable for various biochemical applications.

Conformational preference of 'CαNN' short peptide motif towards recognition of anions.

Among several 'anion binding motifs', the recently described 'C(α)NN' motif occurring in the loop regions preceding a helix, is conserved through evolution both in sequence and its conformation. To establish the significance of the conserved sequence and their intrinsic affinity for anions, a series of peptides containing the naturally occurring 'C(α)NN' motif at the N-terminus of a designed helix, have been modeled and studied in a context free system using computational techniques. Appearance of a single interacting site with negative binding free-energy for both the sulfate and phosphate ions, as evidenced in docking experiments, establishes that the 'C(α)NN' segment has an intrinsic affinity for anions. Molecular Dynamics (MD) simulation studies reveal that interaction with anion triggers a conformational switch from non-helical to helical state at the 'C(α)NN' segment, which extends the length of the anchoring-helix by one turn at the N-terminus. Computational experiments substantiate the significance of sequence/structural context and justify the conserved nature of the 'C(α)NN' sequence for anion recognition through "local" interaction.

Sulfate ion interaction with 'anion recognition' short peptide motif at the N-terminus of an isolated helix: A conformational landscape.

Anion-binding motifs in proteins are typically conserved in sequence and conformation. Crystal structural studies have shown that such motifs often occur in loop regions preceding a helix and interaction with the anions can induce their well defined conformational changes. In order to understand the properties of such motifs in isolation, we have synthesized an 18-residue chimeric polypeptide whose C-terminal part is a designed helix and its N-terminal consists of a C(alpha)NN anion binding structural motif containing residues Leu-Gly-Lys-Gln (residues 107-110 of protein DNA-glycosylase). We present evidence for the interaction of a sulfate (SO(4)(2-)) ion with the L-G-K-Q segment using complementary spectroscopic techniques. Moreover, upon interaction with SO(4)(2-) ion the N-terminal L-G-K-Q segment undergoes a non-helical to helical transition similar to what is observed in protein crystal structure. This work clearly demonstrates the "local" nature of anion binding and the accompanying conformational change that helps in understanding the influence of sequence/structural context of anion binding in proteins.