Determination of soluble and membrane protein structures by X-ray crystallography.

X-ray crystallography is a technique used to determine the atomic-detail structure of a biological macromolecule. The method relies on the ability to generate a three-dimensional crystal of a highly purified protein or nucleic acid for diffraction by X-rays. The extent of scattering of X-rays by the crystal determines the accuracy of the resulting structural model. Unlike electrons, X-rays cannot be refocused after they have been scattered by their target. Thus, calculations are needed to reconstruct the image of the macromolecule that builds the crystal lattice. Tremendous advances over the past 60 years in recombinant expression and purification, crystal growth methods and equipment, X-ray sources, computer processing power, programs, and graphics have taken X-ray crystallography from a highly specialized field to one increasingly accessible to researchers in the biomedical sciences. In this chapter, we review the major concepts of macromolecular X-ray crystallography, focusing mainly on techniques for crystallizing soluble and membrane proteins, and provide a protocol for the crystallization of lysozyme as a model for the crystallization of other proteins.

Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones.

Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228-504) fused to a maltose binding protein (MBP) from E. coli . Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and to ultimately delay the onset of myocilin glaucoma.