nearly 12 copies of GFP on each cubically symmetric scaffold.
After binding GFP to the scaffold, the complex was purified and found by quantitative amino acid analysis to have nearly complete saturation of the binding sites on the scaffold i.e. Taken together, these modifications make a symmetric scaffold that is specific to GFP. Finally, to convert the scaffold to a form that would bind GFP-and thus illustrating the modularity of the system-we exchanged the amino acid sequences in the binding loops on the original DARP14 scaffold with a sequence known as 3G124 19, 20. Motivated by the finding that the terminal repeats of the DARPin were more flexible and showed a compromised resolution, we adopted a set of mutations previously characterized to stabilize this region of the DARPin 23. Compared to the prior study, we modified the DARP14 scaffold as follows. Our current study employed the superfolder variant (sfGFP) 22 with a V206A mutation. GFP has been well-studied and DARPin sequences that bind various forms of GFP have been published 19, 20. In our earlier structural study on this scaffolding system, referred to as DARP14, we analyzed the scaffold by itself without cargo bound and demonstrated that the DARPin adaptor component could be visualized by cryo-EM at a resolution ranging from 3.5 to 5.5 Å 11.Īs a first test case for our scaffolding system, we chose the 26 kDa green fluorescent protein (GFP) as a cargo molecule. With these ideas put together, our scaffold presents multiple symmetrically disposed copies of an adaptor protein whose sequence can be mutated to bind diverse cargo proteins for imaging. In prior work, DARPins have been developed as a facile framework for sequence diversification and selection, by phage display or other laboratory evolution methods, for binding to a wide range of target proteins 14, 15, 16, 17, 18, 19, 20, 21. Our scaffolding system uses a DARPin (Designed Ankyrin Repeat Protein) as an adaptor component that is genetically fused by a continuous alpha helical connection to a central core comprised of a designed symmetric protein cage with cubic symmetry (12 orientations in symmetry T, Fig. In a recent work, we developed a scaffold that addresses the requirements for rigid display and modularity, while also exploiting the advantage of high symmetry to mitigate the common problem of preferred particle orientation in cryo-EM 11.
And to be practical the scaffold must be able to bind diverse cargo proteins with minimal re-engineering efforts.
#Criteria for classification as a protein scaffold full
The cargo protein must be bound and displayed rigidly so that it does not become smeared out during reconstruction of the full structure. Two key challenges have hindered the development of useful cryo-EM scaffolds: rigidity and modularity. molecules of known structure that are large enough to visualize in atomic detail by cryo-EM, and simultaneously able to bind and display a smaller protein molecule of interest, effectively making the smaller ‘cargo’ protein part of a larger assembly that can be structurally elucidated as a whole 8, 9, 10, 11, 12. An alternative approach is to design molecular scaffolding systems-i.e. These studies on small proteins have begun to approach the 38 kDa theoretical lower limit proposed in 1995 3. There, a resolution of 3.2 Å was achieved for human hemoglobin (64 kDa) and streptavidin (52 kDa) 6, 7. In other recent studies, the use of phase plates 5 have been important in enhancing image contrast.
With optimal sample preparations it has been possible in a recent study to reach high resolution (better than 3 Å) for structures as small as 64 kDa 4. The goal of visualizing small proteins by cryo-EM has motivated research efforts along multiple directions. Overcoming this lower size barrier could bring electron microscopy close to the ultimate goal of a universally applicable method for structural biology. 1, 2), a lower size limitation has prevented application of this powerful method to proteins smaller than about 50 kDa 3, which is larger than the average cellular protein. For cryo-EM, despite recent technological advances that have revolutionized the field (reviewed in ref. X-ray crystallography presents difficulties in crystallization, while NMR methods become challenging for very large macromolecules. Yet the leading techniques-X-ray crystallography, NMR, and electron microscopy-all face obstacles that limit their universal application. Methods for visualizing macromolecules in atomic detail have transformed our understanding of molecular biology.
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