Abstract
The development of stable biomolecular scaffolds that can tolerate environmental extremes has considerable potential for industrial and defense-related applications. However, most natural proteins are not sufficiently stable to withstand non-physiological conditions. We have recently engineered the de novo designed Top7 protein to specifically recognize the glycoprotein CD4 by insertion of an eight-residue loop. The engineered variant exhibited remarkable stability under chemical and thermal denaturation conditions. In the present study, far-UV CD spectroscopy and explicit-solvent MD simulations are used to investigate the structural stability of Top7 and the engineered variant under extreme conditions of temperature and pH. Circular dichroism measurements suggest that the engineered variant Top7CB1, like Top7, retains its structure at high temperatures. Changes in CD spectra suggest that there are minor structural rearrangements between neutral and acidic environments for both proteins but that these do not make the proteins less stable at high temperatures. The anti-parallel β-sheet is well conserved within the timescale simulated whereas there is a decrease of helical content when low pH and high-temperature conditions are combined. Concerted alanine mutations along the α-helices of the engineered Top7 variant did not revert this trend when at pH 2 and 400 K. The structural resilience of the anti-parallel β-sheet suggests that the protein scaffold can accommodate varying sequences. The robustness of the Top7 scaffold under extreme conditions of pH and temperature and its amenability to production in inexpensive bacterial expression systems reveal great potential for novel biotechnological applications.
Original language | English |
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Pages (from-to) | 755-765 |
Number of pages | 11 |
Journal | Journal of Molecular Graphics and Modelling |
Volume | 28 |
Issue number | 8 |
DOIs | |
State | Published - Jun 2010 |
Externally published | Yes |
Keywords
- Affinity reagents
- CD4 binding protein
- Chemical and thermal denaturation
- Experimental interpretation
- Protein engineering
- Temperature and pH-dependent conformations
- Thermostable proteins