Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217 and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

ABSTRACTCardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.GRAPHICAL ABSTRACTHIGHLIGHTSA mitochondrial protein-lipid complex regulates lipid peroxidation in apoptosis.Peroxidase-active lipid-cytochrome c complexes are reconstituted in vitro.Phosphatidylglycerol lipids are less effective activators than cardiolipin.Activity correlates to localized dynamics, distinct from chemical denaturation.A dynamic interplay of cytochrome c foldons and anionic lipids regulate activity.

Effect of Stapling on the Thermodynamics of Protein-Peptide Binding

Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. Addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide surprisingly the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.