RESUMO
Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
Assuntos
Encéfalo/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Mutação , Animais , Cristalografia por Raios X , Proteínas de Fluorescência Verde/genética , Ligação de Hidrogênio , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Conformação Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Transdução GenéticaRESUMO
α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α1A-AR and α1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α1A-AR and α1B-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α1A-AR over α1B-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.