Trp-cage miniprotein was used to investigate the role of a salt-bridge (Asp(9) -Arg(16) ) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side-chains are also part of a dense interaction network composed of electrostatic, H-bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt-bridge with those of the salt-bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp-cage miniproteins is more complex than a simple two-state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt-bridge stabilization; in the miniprotein variants equipped with salt-bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt-bridge deleted mutants. In this molecular framework Arg(16) stabilizes more than Asp(9) does, because of its higher degree of 3D-fold cooperation. In conclusion, the Xxx(9) leftright arrow Xxx(16) salt-bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network.

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Exendin-4 (Ex4), a 39-amino acid polypeptide secreted by the lizard Heloderma suspectrum, shares common bioactivity with the human incretin glucagon-like peptide-1 (GLP-1).1 Both Ex4 and GLP-1 bind to the GLP-1 receptor (GLP-1R) and potentiate the secretion of insulin from pancreatic β-cells.2,3 Because of its high binding affinity and extended in vivo half-life, Ex4 and its analogues have been a focus for the treatment of type2 diabetes.4−7The structure of Ex4 has been determined by both 1H NMR spectroscopy and X-ray crystallography.8,9 Because the solubility of Ex4 inH2Ois limited, its NMR spectra have been acquired in a 30% trifluoroethanol (TFE)/70% H2O mixture8 while the X-ray structure of a truncated Ex4 [Ex4(9−39)] has been determined in a GLP-1R-bound state.9 Ex4 displays significant helicity from residue 7 to 28 with greater fraying at the Nterminus and is stabilized by a Trp cage (TC) tertiary fold at the C-terminal part of the helix. The comparison of the NMR and X-ray structures reveals that the TFE/H2O medium can mimic the receptor bound state as the two types of Ex4 conformers superimpose well.The N-terminal segment of Ex4 is 82% homologous with human GLP-1(7−36); conserved residues include His1, Glu3, Thr5, and Phe6, all of which are crucial for receptor activation ( Figure 1).10−12 The affinity of Ex4 for the GLP-1 receptor's N-terminal domain (nGLP-1R) is governed by the central helical part of the molecule.13 Both hydrophobic and hydrophilic side chains are involved in the binding as revealed by the crystal structure of the complex [Protein Data Bank (PDB) entry 3C5T].9 The binding surface of Ex4 consists of Glu15, Val19, Arg20, Phe22, Ile23, Leu26, Lys27, and Ser32, whereas that of nGLP-1R involves Leu32, Thr35, Val36, Trp39, Tyr69, Tyr88, Leu89, Pro90, Trp91, Leu123, Glu127, and Glu128.9On the basis of recent structure−activity studies, it is known that all peptidic ligands targeting class B G-protein-coupled receptors (GPCRs) bind in a predominantly α-helical manner, and for most ligands, the α-helix forms upon binding of the extracellular N-terminal domain of the GPCR.15 These ligands, including glucagon,16 GLP-1,8,17 GLP-2,18 Ex4,8 glucose dependent insulinotropic polypeptide (GIP),19 pituitary adenylate cyclase activating polypeptide (PACAP),20 and corticotropin-releasing factor (CRF),21 show moderately ordered structure in aqueous solution but adopt a continuous or kinked α-helix in the presence of organic solvents (e.g., in a TFE/water mixed solvent) or in protein crystals. The major structural difference between Ex4 and GLP-1 is the continuity of the α-helix: Ex4 forms a single amphipathic α-helix both in the TFE/water mixed solvent and in the receptor-bound state, while GLP-1 is composed of two adjacent subhelices joined by the flexible Gly22. Binding studies demonstrated that the isolated N-terminal extracellular domain of GLP-1R has a low affinity for endogenous GLP-1 but a high affinity for Ex4.22 The superior affinity of Ex4 stems from the more favorable alignment of the oppositel...