Alanine-shaving mutagenesis to determine key interfacial residues governing the assembly of a nano-cage maxi-ferritin.
The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperature-dependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nano-structures but can also help to understand the role that structure plays on the function of this important class of proteins.
Zhang
Yu
Y
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore.
Raudah
Siti
S
Teo
Huihian
H
Teo
Gwenda W S
GW
Fan
Rongli
R
Sun
Xiaoming
X
Orner
Brendan P
BP
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D005798:Genes, Bacterial; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D016296:Mutagenesis; D049329:Nanostructures / Q000737:chemistry / Q000648:ultrastructure; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005293:Ferritins; C018032:bacterioferritin