Lisa A. Eidenschink

Stabilizing capping motif for β-hairpins and sheets

Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most β-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, “unnatural” beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 + kJ/mol to β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands and reduces fraying at the ends of hairpins and other β-sheet models. Utilizing this motif, 10- to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The β-cap can also be used to staple together short antiparallel β-strands connected by a long flexible loop.

Very short peptides with stable folds: Building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone–amide interaction geometries

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C‐terminal H‐bonding interaction between a backbone amide and an i‐2 Trp ring, a particularly stable (ΔGU > 7 kJ/mol) truncated hairpin, Ac‐WI‐(D‐Pro‐D‐Asn)‐KWTG‐NH2, results. In this construct and others with a W‐(4‐residue turn)‐W motif in severely truncated hairpins, the C‐terminal Trp is the edge residue in a well‐defined face‐to‐edge (FtE) aryl/aryl interaction. Longer hairpins and those with six‐residue turns retain the reversed “edge‐to‐face” (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W‐(4‐residue turn)‐W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater β‐propensity of Trp. The π–cation, and Trp/Gly‐HN interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly‐HN in ‐WXnG‐ units both in turns (n = 2) and at the C‐termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred “face” residue in aryl/aryl pairings, presumably because of its greater π basicity. Proteins 2009.

Terminal sidechain packing of a designed β-hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔGU = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster.

Development of Calcitonin Salmon Nasal Spray: Similarity of Peptide Formulated in Chlorobutanol Compared to Benzalkonium Chloride as Preservative

The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miacalcin Nasal Spray). Various orthogonal methods assessed peptide structuring, dynamics, and aggregation state. Mass spectrometry, amino acid analysis, and N-terminal sequencing all demonstrated similarity in primary structure. Near- and far-UV circular dichroism (CD) data supported similarity in secondary and tertiary sCT structure. Nuclear magnetic resonance studies further supported similarity of three-dimensional structure and molecular dynamics of the peptide. Other methods, such as sedimentation velocity and size exclusion chromatography, demonstrated similarity in peptide aggregation state. These latter methods, in addition to reversed phase chromatography, were also employed for monitoring stability under forced degradation, and at the end of recommended shelf storage and patient use conditions. In all cases and for all methodologies employed, similarity to the RLD was observed with respect to extent of aggregation and other degradation processes. Finally, ELISA and bioassay data demonstrated similarity in biological properties. These investigations comprehensively demonstrate physicochemical similarity of Calcitonin Salmon Nasal Spray and the RLD, and should prove a useful illustration to pharmaceutical scientists developing alternative and/or generic peptide or protein products.

Aryl‐Aryl interactions in designed peptide folds: Spectroscopic characteristics and placement issues for optimal structure stabilization

We have extended our studies of Trp/Trp to other Aryl/Aryl through‐space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T‐shape orientation. Designated as an edge‐to‐face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W ≫ Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225–232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197–200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp‐cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross‐strand aryl/aryl clusters for the design of stable β‐sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts.

Aryl-aryl interactions in designed peptide folds: Spectroscopic characteristics and optimal placement for structure stabilization: Aryl-Aryl Interactions in Designed Peptide Folds

We have extended our studies of Trp/Trp to other Aryl/Aryl through‐space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T‐shape orientation. Designated as an edge‐to‐face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W ≫ Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225–232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197–200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp‐cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross‐strand aryl/aryl clusters for the design of stable β‐sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts.

Determinants of fold stabilizing aromatic-aromatic interactions in short peptides.

The significance of interactions involving aromatic side-chains in stabilizing protein structure is well accepted, while the geometry and specificity of these interactions are more elusive. Hydrophobic clustering plays a significant role; these interactions can be distinguished as aromatic/aliphatic and aromatic/aromatic interactions, with the aromatic/aromatic interactions displaying two dominant geometries–edge-to-face (EtF) and parallel displaced (PD) stacking [1]. Cross-strand aryl/aryl pairings occur predominantly at non-H-bonded sites in β-sheets [2]. In β-hairpin models these have been found to be stabilizing at turn flanking positions [3]. The excised N-terminal hairpin of the B1 domain of Protein G has a Tyr/Phe pair at such a position and is required for hairpin formation [4]. Trpzip4 and its analogs display two EtF Trp/Trp interactions at non-H-bonded sites, the turn flanking pair accounts for the majority of stabilization. HP6 and HP7, two peptide series more remotely related to GB1, have also shown remarkable stability attributed to a turn flanking EtF Trp/Trp pair. In all of its incarnations the interaction has been seen to maintain a specific geometry, with the edge of the N-terminal Trp abutting the face of the C-terminal Trp. The stability of HP7 and its truncated version, as well as chignolin [5], suggest that an EtF aromatic/aromatic interaction immediately flanking a turn sequence is particularly stabilizing in small β-hairpins. HP6V has the same stabilizing EtF interaction but a β-turn that is less favorable in systems with short β-strands, provided an excellent system to test the limit of the W/W EtF interaction. The first truncated peptide of this design, AW-SNGK-WT, displayed the usual CD exciton couplet, in fact larger than expected, and an upfield Trp He3 with a melting curve suggesting a Tm of circa 25°C, apparently more stable than Ac-WNPATGKW-NH2, the 8-mer with the optimized reversing loop. NOESY sequencing, however, indicated that the upfield He3 signal was in the C-terminal, rather than N-terminal Trp. A battery of small Trp containing peptides was examined to ascertain the determinants of the EtF geometry between the Trp sidechains. The transition was not found to possess an absolute boundary; a “middle ground” with two folded states corresponding to the two EtF geometries was observed. A 2.7 ppm upfield shift (CSD) seen for the G8-HN of Ac-WTNGKWTG-NH2 (peptide WP) suggested a local aryl-amide interaction at the N-terminus. Aryl-X-Gly i → i+2 interactions can act as modest structuring elements even in denatured proteins [6] and peptides, evidenced by Gly HN CSDs up to −1.4. Only in proteins have Aryl-XG shifts as large as that in peptide WP been observed.

Optimization of a β-sheet-cap for long loop closure.

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: RWITVTI – loop – KKIRVWE). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RWITVTI‐(GGGGKK)2GGGG‐KKIRVWE) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RWITVRIW‐(GGGGKK)2GGGG‐WKTIRVWE) supports a remarkably stable β‐sheet (ΔGU = 6.3 kJ/mol at 295 K, Tm = ∼55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.