Structural water stabilizes protein motifs in liquid protein phase: The folding mechanism of short β-sheets coupled to phase transition

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small β-sheets, the key motifs of proteins, undergoing structural changes in liquid–liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3–0.5 kcal\ub7mol−1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2–2.3 kcal\ub7mol−1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal\ub7mol−1 per residue excess stabilization

Chiral 1,5-disubstituted 1,2,3-triazoles-versatile tools for foldamers and peptidomimetic applications

1,4- A nd 1,5-Disubstituted triazole amino acid monomers have gained increasing interest among peptidic foldamers, as they are easily prepared via Cu- A nd Ru-catalyzed click reactions, with the potential for side chain variation. While the latter is key to their applicability, the synthesis and structural properties of the chiral mono-or disubstituted triazole amino acids have only been partially addressed. We here present the synthesis of all eight possible chiral derivatives of a triazole monomer prepared via a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC). To evaluate the conformational properties of the individual building units, a systematic quantum chemical study was performed on all monomers, indicating their capacity to form several low energy conformers. This feature may be used to effect structural diversity when the monomers are inserted into various peptide sequences. We envisage that these results will facilitate new applications for these artificial oligomeric compounds in diverse areas, ranging from pharmaceutics to biotechnology

Stimuli-Responsive Membrane Anchor Peptide Nanofoils for Tunable Membrane Association and Lipid Bilayer Fusion

Self-assembled peptide nanostructures with stimuli-responsive features are promising as functional materials. Despite extensive research efforts, water-soluble supramolecular constructs that can interact with lipid membranes in a controllable way are still challenging to achieve. Here, we have employed a short membrane anchor protein motif (GLFD) and coupled it to a spiropyran photoswitch. Under physiological conditions, these conjugates assemble into ∼3.5 nm thick, foil-like peptide bilayer morphologies. Photoisomerization from the closed spiro (SP) form to the open merocyanine (MC) form of the photoswitch triggers rearrangements within the foils. This results in substantial changes in their membrane-binding properties, which also varies sensitively to lipid composition, ranging from reversible nanofoil reformation to stepwise membrane adsorption. The formed peptide layers in the assembly are also able to attach to various liposomes with different surface charges, enabling the fusion of their lipid bilayers. Here, SP-to-MC conversion can be used both to trigger and to modulate the liposome fusion efficiency

Michler's Hydrol Blue: A Sensitive Probe for Amyloid Fibril Detection

Michlers hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14 degrees-22 degrees to the fibril axis and along the grooves of the beta-sheet

Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

Despite exhaustive chemical and crystal structure studies, the mechanistic details of how F o F 1 -ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO 3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in β TP and, thus, promote ATP. \ua9 2009 American Chemical Society

Energy phase shift as mechanism for catalysis

Catalysts are agents that by binding reactant molecules lower the energy barriers to chemical reaction. After reaction the catalyst is regenerated, its unbinding energy recruited from the environment, which is associated with an inevitable loss of energy. We show that combining several catalytic sites to become energetically and temporally phase-shifted relative to each other provides a possibility to sustain the overall reaction by internal 'energy recycling', bypassing the need for thermal activation, and in principle allowing the system to work adiabatically. Using an analytical model for superimposed, phase-shifted potentials of F-1-ATP synthase provides a description integrating main characteristics of this rotary enzyme complex

High anisotropy of flow-aligned bicellar membrane systems

In recent years, multi-lipid bicellar systems have emerged as promising membrane models. The fast orientational diffusion and magnetic alignability made these systems very attractive for NMR investigations. However, their alignment was so far achieved with a strong magnetic field, which limited their use with other methods that require macroscopic orientation. Recently, it was shown that bicelles could be aligned also by shear flow in a Couette flow cell, making it applicable to structural and biophysical studies by polarized light spectroscopy. Considering the sensitivity of this lipid system to small variations in composition and physicochemical parameters, efficient use of such a flow-cell method with coupled techniques will critically depend on the detailed understanding of how the lipid systems behave under flow conditions. In the present study we have characterized the flow alignment behavior of the commonly used dimyristoyl phosphatidylcholine/dicaproyl phosphatidylcholine (DMPC/DHPC) bicelle system, for various temperatures, lipid compositions, and lipid concentrations. We conclude that at optimal flow conditions the selected bicellar systems can produce the most efficient flow alignment out of any lipid systems used so far. The highest degree of orientation of DMPC/DHPC samples is noticed in a narrow temperature interval, at a practical temperature around 25 C, most likely in the phase transition region characterized by maximum sample viscosity. The change of macroscopic orientation factor as function of the above conditions is now described in detail. The increase in macroscopic alignment observed for bicelles will most likely allow recording of higher resolution spectra on membrane systems, which provide deeper structural insight and analysis into properties of biomolecules interacting with solution phase lipid membranes. (C) 2013 Elsevier Ireland Ltd. All rights reserved

Ruthenium-Catalyzed Azide Alkyne Cycloaddition (RuAAC)

Under ruthenium catalysis, 1,5-disubstituted 1,2,3-triazoles can be accessed with high selectivity from terminal alkynes and organic azides via a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) reaction. In addition, these conditions allow the use of internal alkynes, providing access to 1,4,5-trisubstituted 1,2,3-triazoles. This chapter reviews the scope and limitations of the RuAAC reaction, as well as selected applications. A brief mention of AAC reaction catalyzed by other metals is also included

Conformational properties of 1,4- and 1,5-substituted 1,2,3-triazole amino acids-building units for peptidic foldamers

Peptidic foldamers have recently emerged as a novel class of artificial oligomers with properties and structural diversity similar to that of natural peptides, but possessing additional interesting features granting them great potential for applications in fields from nanotechnology to pharmaceuticals. Among these, foldamers containing 1,4- and 1,5-substitued triazole amino acids are easily prepared via the Cu- and Ru-catalyzed click reactions and may offer increased side chain variation, but their structural capabilities have not yet been widely explored. We here describe a systematic analysis of the conformational space of the two most important basic units, the 1,4-substitued (4Tzl) and the 1,5-substitued (5Tzl) 1,2,3-triazole amino acids, using quantum chemical calculations and NMR spectroscopy. Possible conformations of the two triazoles were scanned and their potential minima were located using several theoretical approaches (B3LYP/6-311++G(2d,2p), ωB97X-D/6-311++G(2d,2p), M06-2X/6-311++G(2d,2p) and MP2/6-311++G(2d,2p)) in different solvents. BOC-protected versions of 4Tzl and 5Tzl were also prepared via one step transformations and analyzed by 2D NOESY NMR. Theoretical results show 9 conformers for 5Tzl derivatives with relative energies lying close to each other, which may lead to a great structural diversity. NMR analysis also indicates that conformers preferring turn, helix and zig-zag secondary structures may coexist in solution. In contrast, 4Tzl has a much lower number of conformers, only 4, and these lack strong intraresidual interactions. This is again supported by NMR suggesting the presence of both extended and bent conformers. The structural information provided on these building units could be employed in future design of triazole foldamers