Herein,
we report a conjugation strategy, where we utilize a poly(ethylene
oxide) cylindrical molecular brush architecture to design a self-assembled
structure for thermal stabilization of enzymes. We demonstrate that
the proposed architecture of the moderately stiff polymer ligand results
in a significant improvement of biocatalytic activity and thermal
stability of lysozyme and trypsin that retain their activity, even
upon heating to 100 °C and above. The molecular brush is bound
via epoxy functional groups to the amino groups of the lysine on the
surface of the enzyme globule, promoting the formation of stiff and
crowded cages around the enzymes and preventing the water molecules
access to the enzyme and enzymes agglomeration. The molecular dynamic
simulations show that the high concentration of poly(ethylene oxide)
in the vicinity of the enzyme is critical for their stability. Monitoring
of lysozyme–molecular brush conjugates for 6 and 12 months
in lyophilized form and in solution, respectively, has shown that
the conjugation does not compromise the shelf life of the enzyme.
Amyloid
fibrils are large aggregates of misfolded proteins, which
are often associated with various neurodegenerative diseases such
as Alzheimer’s, Parkinson’s, Huntington’s, and
vascular dementia. The amount of hydrogen sulfide (H2S)
is known to be significantly reduced in the brain tissue of people
diagnosed with Alzheimer’s disease relative to that of healthy
individuals. These findings prompted us to investigate the effects
of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL).
HEWL forms typical β-sheet rich fibrils during the course of
70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of β-sheet and amyloid
fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy
and ThT fluorescence. Nonresonance Raman spectroscopy shows that disulfide
bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational
modes in the 550–500 cm–1 spectral range
decrease in intensity and are accompanied by the appearance of a new
490 cm–1 band assigned to the trisulfide group (RSSSR)
based on the comparison with model compounds. The formation of RSSSR
was proven further using a reaction with TCEP reduction agent and
LC-MS analysis of the products. Intrinsic tryptophan fluorescence
study shows a strong denaturation of HEWL containing trisulfide bonds.
The presented evidence indicates that H2S causes the formation
of trisulfide bridges, which destabilizes HEWL structure, preventing
protein fibrillation. As a result, small spherical aggregates of unordered
protein form, which exhibit no cytotoxicity by contrast with HEWL
fibrils.
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