muramidase and tris(2-carboxyethyl)phosphine

Amyloid formation of proteins is not only relevant for neurodegenerative diseases, but has recently emerged as a groundbreaking approach in materials science and biotechnology. However, amyloid aggregation of proteins in vitro generally requires a long incubation time under extremely harsh conditions, and the understanding of the structural motif to determine amyloid assembly is extremely limited. Herein we reveal that the integration of three important building blocks in typical globular proteins is crucial for superfast protein amyloid-like assembly including the segment required for high fibrillation propensity, abundant α-helix structures and intramolecular S-S bonds to lock the α-helix. With the reduction of the S-S bond by tris(2-carboxyethyl)phosphine (TCEP), the α-helix was rapidly unlocked from the protein chain, and the resultant unfolded monomer underwent a fast transition to β-sheet-rich amyloid oligomers and protofibrils in minutes, which further assembled into a macroscopic nanofilm at the air/water interface and microparticles in bulk solution, respectively.

Topics: Amino Acid Motifs; Amyloid; Disulfides; Muramidase; Nanostructures; Phosphines

Macromolecular crystallization has many implications in biological and materials science. Similar to the crystallization of other molecules, macromolecular crystallization conventionally considers a critical nucleus, followed by crystallographic packing of macromolecules to drive further crystal growth. Herein, we discover a distinctive macromolecular crystallization pathway by developing the concept of a macromolecular mesocrystal. This nonclassical polymer crystallization occurs through the mesoscale self-assembly of (bio)macromolecular nanocrystals. The new concept for macromolecular crystallization presented herein is fundamental and relevant to many fields, including materials science, chemistry, biomimetics, nanoscience, and structural biology.

Topics: Crystallization; Hydrogen-Ion Concentration; Macromolecular Substances; Muramidase; Nanoparticles; Phosphines; Protein Unfolding; Spectrum Analysis, Raman; Temperature

Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.

Topics: Anilino Naphthalenesulfonates; Animals; Cattle; Chickens; Disulfides; Dithiothreitol; Microscopy, Electron, Scanning; Muramidase; Oxidation-Reduction; Phosphines; Protein Denaturation; Protein Structure, Tertiary; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet

Gold nanoparticles are useful in biomedical applications due to their distinct optical properties and high chemical stability. Reports of the biogenic formation of gold colloids from gold complexes has also led to an increased level of interest in the biomineralization of gold. However, the mechanism responsible for biomolecule-directed gold nanoparticle formation remains unclear due to the lack of structural information about biological systems and the fast kinetics of biomimetic chemical systems in solution. Here we show that intact single crystals of lysozyme can be used to study the time-dependent, protein-directed growth of gold nanoparticles. The protein crystals slow down the growth of the gold nanoparticles, allowing detailed kinetic studies to be carried out, and permit a three-dimensional structural characterization that would be difficult to achieve in solution. Furthermore, we show that additional chemical species can be used to fine-tune the growth rate of the gold nanoparticles.

Topics: Crystallization; Crystallography, X-Ray; Gold; Kinetics; Mercury; Metal Nanoparticles; Microscopy, Electron, Transmission; Models, Molecular; Muramidase; Particle Size; Phosphines; Time Factors; Tomography

This work examines the inhibitory effect of TCEP on the in vitro fibrillation of hen lysozyme at pH 2. We demonstrate that the inhibition of hen lysozyme fibrillation by TCEP follows a dose-dependent manner. Our data show that the addition of TCEP prevents alpha-to-beta transition and promoted unfolding of lysozyme. Moreover, our findings suggested that the TCEP-induced attenuated fibrillation is associated with disulfide disruption and structural unfolding of HEWL.

Topics: Amyloid; Animals; Hydrogen-Ion Concentration; Microscopy, Electron; Muramidase; Phosphines; Protein Structure, Secondary

The role of protein structure in the reactivity of the four disulfide (S-S) bridges of lysozyme was studied using Raman spectroscopy and molecular modelling. The experimental kinetics of S-S bridge reduction by tris-2-carboxyethyl phosphine (TCEP) was obtained by monitoring the protein S-S Raman bands. The kinetics are heterogeneous and were fitted using two apparent reaction rate constants. Kinetic measurements performed at different pH values indicate only moderate charge effects. The two intrinsic reaction rate constants derived for the neutral TCEP species were 0.45 and 0.052 mol(-1) s(-1), respectively. The molecular dynamics simulation of the reactants encounter shows that the accessibility of the lysozyme S-S bridges by TCEP decreases in the following order: cys30-cys115 > cys6-cys127 > cys64-cys80 > cys76-cys94. This simulation also illustrates the reaction mechanism which consists of a local unfolding followed by the reduction of the exposed S-S bridge. The Gibbs free energy for local unfolding was evaluated by comparing the actual reaction rate constant with that of a model system containing a fully exposed S-S bridge (oxidized glutathione). These values corresponding to the fast- and slow-reaction rate-constants were 8.5 and 13.8 kJ mol(-1), respectively. On the other hand, Raman measurements, as well as the molecular dynamics simulations, strongly suggest that the protein global unfolding following S-S bridge cleavage has only limited effects in stabilizing the reaction products.

Topics: Animals; Computer Simulation; Disulfides; Kinetics; Models, Molecular; Muramidase; Phosphines; Protein Binding; Protein Conformation; Quantum Theory; Reducing Agents; Spectrum Analysis, Raman; Thermodynamics