Sequence, structure, and cooperativity in folding of elementary protein structural motifs.

Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

Site-specific thermodynamic stability and unfolding of a de novo designed protein structural motif mapped by 13C isotopically edited IR spectroscopy.

The mechanism of protein folding remains poorly understood, in part due to limited experimental information available about partially folded states. Isotopically edited infrared (IR) spectroscopy has emerged as a promising method for studying protein structural changes with site-specific resolution, but its full potential to systematically probe folding at multiple protein sites has not yet been realized. We have used (13)C isotopically edited IR spectroscopy to investigate the site-specific thermal unfolding at seven different locations in the de novo designed helix-turn-helix protein αtα. As one of the few stable helix-turn-helix motifs, αtα is an excellent model for studying the roles of secondary and tertiary interactions in folding. Circular dichroism (CD) experiments on the full αtα motif and its two peptide fragments show that interhelical tertiary contacts are critical for stabilization of the secondary structure. The site-specific thermal unfolding probed by (13)C isotopically edited IR is likewise consistent with primarily tertiary stabilization of the local structure. The least thermally stable part of the αtα motif is near the turn where the interhelical contacts are rather loose, while the motif's center with best established core packing has the highest stability. Similar correlation between the local thermal stability and tertiary contacts was found previously for a naturally occurring helix-turn-helix motif. These results underline the importance of native-like tertiary stabilizing interactions in folding, in agreement with recent state-of-the art folding simulations as well as simplified, native-centric models.

Multi-Probe Equilibrium Analysis of Gradual (Un)Folding Processes.

Studies of small proteins that exhibit noncooperative, gradual (un)folding can offer unique insights into the rarely accessible intermediate stages of the protein folding processes. Detailed experimental characterization of these intermediate states requires approaches that utilize multiple site-specific probes of the local structure. Isotopically edited infrared (IR) spectroscopy has emerged as a powerful methodology capable of providing such high-resolution structural information. Labeling of selected amide carbonyls with 13C results in detectable side-bands of amide I' vibrations, which are sensitive to local conformation and/or solvent exposure without introducing any significant structural perturbation to the protein. Incorporation of isotopically labeled amino acids at specific positions can be achieved by the chemical synthesis of the studied proteins. We describe the basic procedures for synthesis of 13C isotopically edited protein samples, experimental IR spectroscopic measurements and analysis of the site-specific equilibrium thermal unfolding of a small protein from the temperature-dependent IR data.

Effect of Mutations on the Global and Site-Specific Stability and Folding of an Elementary Protein Structural Motif.

Understanding the folding mechanism of proteins requires detailed knowledge of the roles of individual amino acid residues in stabilization of specific elements and local segments of the native structure. Recently, we have utilized the combination of circular dichroism (CD) and site-specific 13C isotopically edited infrared spectroscopy (IR) coupled with the Ising-like model for protein folding to map the thermal unfolding at the residue level of a de novo designed helix-turn-helix motif αtα. Here we use the same methodology to study how the sequence of local thermal unfolding is affected by selected mutations introduced into the most and least stable parts of the motif. Seven different mutants of αtα are screened to find substitutions with the most pronounced effects on the overall stability. Subsequently, thermal unfolding of two mutated αtα sequences is studied with site-specific resolution, using four distinct 13C isotopologues of each. The data are analyzed with the Ising-like model, which builds on a previous parametrization for the original αtα sequence and tests different ways of incorporating the amino acid substitution. We show that for both more and less stable mutants only the adjustment of all interaction parameters of the model can yield a satisfactory fit to the experimental data. The stabilizing and destabilizing mutations result, respectively, in a similar increase and decrease of the stability of all probed local segments, irrespective of their position with respect to the mutation site. Consequently, the relative order of their unfolding remains essentially unchanged. These results underline the importance of the interconnectivity of the stabilizing interaction network and cooperativity of the protein structure, which is evident even in a small motif with apparently noncooperative, heterogeneous unfolding. Overall, our findings are consistent with the native structure being the dominant factor in determining the folding mechanism, regardless of the details of its overall or local thermodynamic stabilization.