JAN STEYAERT
Nanobody-Enabled Structural Biology
Published research of the Steyaert lab (see www.steyaertlab.eu for all details) established that the small and rigid recombinant antigen binding fragments (15kDa) of Camelid heavy chain only antibodies - known as Nanobodies - constitute unique research tools in structural biology. By rigidifying flexible regions and obscuring aggregative surfaces, Xaperone complexes warrant conformationally uniform samples that are key to protein structure determination by X-ray crystallography. Xaperones (Xaps) are antigenbinding fragments from heavy-chain-only antibodies that are able to:
• bind cryptic epitopes and lock proteins in unique native conformations
• increase the stability of soluble proteins and solubilized membrane proteins
• reduce the conformational complexity of soluble proteins and solubilized membrane proteins
• increase the polar surface enabling the growth of diffracting crystal
The useful applications of Xaperones in structural biology are numerous. Xaperones increase the hydrophilic surface of integral membrane proteins and reduce their conformational heterogeneity. Xaperones can trap unstable structural intermediates along the fibrillation pathway of amyloidogenic proteins. A multidomain protein is more rigid in a complex with a Xaperone than the multidomain protein by itself. In complex with a Xaperone, the total amount of structured polypeptide increases, thus providing a much better starting point for the crystallization of intrinsically unfolded proteins. Xaperones are suitable to stabilize the protomers of larger protein assemblies in one-to-one heterodimers or transient protein protein complexes.
Recent highlights include the elucidation of the first GPCR structure in its active state using a conformational selective Nanobody (Nature News and views) and the structural investigation of the GPCR-G protein complex by Xaperone-assisted X-ray crystallography (Nature News and views).
• bind cryptic epitopes and lock proteins in unique native conformations
• increase the stability of soluble proteins and solubilized membrane proteins
• reduce the conformational complexity of soluble proteins and solubilized membrane proteins
• increase the polar surface enabling the growth of diffracting crystal
The useful applications of Xaperones in structural biology are numerous. Xaperones increase the hydrophilic surface of integral membrane proteins and reduce their conformational heterogeneity. Xaperones can trap unstable structural intermediates along the fibrillation pathway of amyloidogenic proteins. A multidomain protein is more rigid in a complex with a Xaperone than the multidomain protein by itself. In complex with a Xaperone, the total amount of structured polypeptide increases, thus providing a much better starting point for the crystallization of intrinsically unfolded proteins. Xaperones are suitable to stabilize the protomers of larger protein assemblies in one-to-one heterodimers or transient protein protein complexes.Recent highlights include the elucidation of the first GPCR structure in its active state using a conformational selective Nanobody (Nature News and views) and the structural investigation of the GPCR-G protein complex by Xaperone-assisted X-ray crystallography (Nature News and views).
- A general protocol for the generation of Nanobodies for structural biology. (Nat Protoc, 9, 674-93, 2014)
- Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody. (J Am Chem Soc, 136, 937-44, 2014)
- Conformational biosensors reveal GPCR signalling from endosomes. (Nature, 495, 534-8, 2013)
- Crystal structure of the β2 adrenergic receptor-Gs protein complex. (Nature, 477, 549-55, 2011)
- Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. (Nature, 469, 175-80, 2011)
