The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation

Abstract

The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal (La 3؉ ) binding. The heat of La 3؉ interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a La 3؉ -binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (i) the enthalpy of helix formation is ؊0.9 ؎ 0.1 kcal͞mol; (ii) the enthalpy of helix formation is independent of the peptide length; (iii) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45°C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of ␣-helices. The year 2001 marked the 50th anniversary of the ␣-helix, the first proposed protein secondary structure (1). However, despite numerous efforts (see refs. 2-5 and references therein), the detailed thermodynamic basis for helix formation and for the helix propensities of the amino acids is not yet well understood. Accurate values for such basic thermodynamic parameters as the changes in enthalpy and heat capacity on helix formation are still under debate. Recently Bierzynski and coworkers (6, 7) developed a peptide system for inducing helix formation by adding a metal ion. They took a 12-residue sequence, analogous to a Ca 2ϩ -binding loop from calmodulin (peptide P1), which forms a short and very stable C-terminal helix, containing three to four residues, when the peptide binds La 3ϩ . The C-terminal segment of La 3ϩ -bound P1 provides a stable helical nucleus for helix propagation when additional residues are added at the C terminus. Bierzynski and coworkers also made a longer peptide (P2) with three additional alanine residues plus one C-terminal glutamine residue, and determined the NMR structure of the La 3ϩ -ligated form. Their model system should allow accurate determination of the enthalpy of helix formation by using ITC (isothermal titration calorimetry) to monitor stepwise, La 3ϩ -induced helix formation, and we exploit this potential here.Helix-coil transitions of short peptides in water cannot usually be described by a two-state model because the helix propagation constant is not large enough. For this reason, and also because the enthalpy change on helix formation is not large, temperatureinduced helix unfolding spans a very broad temperature range, which makes it difficult to determine the baseline. Analysis of thermal helix-coil transitions can be based on the Zimm-Bragg or Lifson-Roig theories (e.g., see ref.2), but the enthalpy values determined in t...

End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation

“…Thus, the only possible intrachain interactions in our model chains are hydrogen bonds between backbone amide and backbone carbonyl groups or between backbone carbonyl groups and the serine side chain. Our results indicate that these intramolecular hydrogen bonds are more favorable than hydrogen bonds between the peptide chain and water, which may be due to a favorable solvation free energy of solvent-exposed intramolecular hydrogen bonds, which was recently found to be the major determinant for the stability of alanine-based ␣-helices (38)(39)(40). The presence of intramolecular hydrogen bonds in unfolded proteins has major consequences for the contributions of hydrogen bonds to protein stability.…”

Length Dependent Helix−Coil Transition Kinetics of Nine Alanine-Based Peptides

“…It has been well established that the enthalpy of helix formation amounts to approximately 1.0 kcal/mol per residue. 60 Thus, thermal unfolding curves of monomeric helices become increasingly steep with increasing chain length, although the van't Hoff enthalpy of the relaxation never reaches the calorimetric enthalpy due to the multistate nature of the transition as well as contributions from both folding and unfolding.…”

Inhibitory mechanism of peptides with a repeating hydrophobic and hydrophilic residue pattern on interleukin-10

“…52,53 In P2.2 and P2.3, we used 'WY' and 'FY' to substitute 'FF' in the first and second 'FFRR' units, respectively, to make the N-terminus less hydrophobic (but still hydrophobic overall). Additionally, stabilizing the last helix was facilitated by 2 'AH' substitutions of 'FF', as Ala is able to significantly stabilize helices in short peptides [54][55][56] and His has been found to increase the stability of helices by Hbonding. 57,58 Moreover, in P2.3, we replaced 'RR' by 'HH' in the first 2 'XXRR' (X represents an amino acid) units, which would also stabilize the first helix at physiological pH 57 ; additionally, it has been reported that solvent-exposed intra-helical Trp and His bridges could increase the helical conformation.…”