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Thermostability and molecular encapsulation within an engineered caged protein scaffold
Author(s) -
Dalmau Mercè,
Lim Sierin,
Chen Helen C.,
Ruiz Cesar,
Wang SzuWen
Publication year - 2008
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21988
Subject(s) - thermostability , scaffold , scaffold protein , biophysics , chemistry , mutant , circular dichroism , protein engineering , crystallography , nanotechnology , materials science , biochemistry , biology , enzyme , medicine , signal transduction , gene , biomedical engineering
Self‐assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli . The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 ± 0.9°C and an apparent midpoint unfolding temperature of 91.4 ± 1.4°C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild‐type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non‐native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications. Biotechnol. Bioeng. 2008;101: 654–664. © 2008 Wiley Periodicals, Inc.