Refolding kinetics of lysozyme:

Baloğlu, Çetin (2005) Refolding kinetics of lysozyme:. [Thesis]

[thumbnail of baloglucetin.pdf] PDF

Download (1MB)


NMR spectral analyses and molecular dynamics (MD) simulations in the temperature range of 300-355K and at 500K were used to probe the unfolding, refolding and gelation of native hen egg-white lysozyme. In the first part, preliminary ¹H experiments were conducted on samples that had been freshly dissolved in 10%D₂O / 90%H₂O and spiked with acetonitrile as reference. The samples were encouraged to unfold and refold by ramping the temperature of the NMR probe, yielding real-time spectra that reflected changes of structure. NMR experiments were also performed on lysozyme that had been labeled along the surface with isotopically enriched ¹³C-methyl groups. This strategy, which promoted carbon observation at sensitivities approaching that of many homonuclear ¹H experiments, facilitated the investigation of heteronuclear Nuclear Overhauser effects, spin-lattice (T₁) and spin-spin (T₂) ¹³C relaxation times in the modified protein. Furthermore, the thermal gelation of egg-white lysozyme was monitored in solution containing different amounts of vitamin B₁ as model incipient. Protein incubated with moderate amounts of vitamin B₁, an established enzyme cofactor, showed higher tolerance to the denaturing effects of elevated temperature. In comparison, MD simulations were used to characterize the global changes of protein structure upon thermal treatment and served as a knowledge base to carry out subsequent NMR backbone dynamics studies. The backbone dynamics of lysozyme were assessed using 2ns MD simulations between 300K 355K and 4ns for 500K, in which C fluctuation vector, relaxation times, heat capacity, accessible surface area and solution scattering functions were compared against related experimental findings. In general, the results supported previous interpretations that a protein fingerprint exists, which make out distinct intermediates forming along the unfolding pathways.
Item Type: Thesis
Uncontrolled Keywords: Hen Egg-White Lysozyme. -- Blue shows basic residues. -- Red acidic. -- Green polar. -- White non-polar
Subjects: Q Science > QD Chemistry
Divisions: Faculty of Engineering and Natural Sciences > Basic Sciences > Chemistry
Faculty of Engineering and Natural Sciences
Depositing User: IC-Cataloging
Date Deposited: 15 Apr 2008 10:03
Last Modified: 26 Apr 2022 09:44

Actions (login required)

View Item
View Item