NMR methods for analysis of CRALBP retinoid binding

Abstract

Publisher Summary The identification of the cellular retinaldehyde-binding protein (CRALBP) retinoid-binding pocket and definition of the structural properties of the protein that provide high ligand stereoselectivity and low photosensitivity has been a prime matter of interest. In this purpose, solution state nuclear magnetic resonance (NMR) analysis has been initiated using human recombinant CRALBP labeled by biosynthetic isotope incorporation. A combination of heteronuclear gradient-enhanced 15N NMR and one-dimensional 19F and 13C NMR methods coupled with improved isotope incorporation methods and mass spectrometry, has proven to be complimentary approaches for characterizing CRALBP-ligand interactions. While these methods have been used separately elsewhere, usually as a primary approach to structural problems, this chapter emphasizes the complimentarity of the techniques and the advantages of combining the methods for studying protein–ligand interactions. Such protein biotechnology is suitable for characterizing a variety of protein–igand interactions and is becoming more accessible through specialized biomolecular resource facilities. This study describes the applicability of 15N, 19F, and 13C NMR methodology for studying ligand interactions in a light sensitive protein, such as recombinant CRALBP (rCRALBP). Gradient enhanced sensitivity enhanced heteronuclear single quantum correlation 15N NMR has provided evidence that rCRALBP undergoes a specific localized conformational change upon photoisomerization of 11-cis-retinaldehyde and removal of the ligand from the binding pocket. The results from the multidimensional NMR measurements strongly support the likelihood that the 19F Trp and 13C-Met NMR chemical shift differences observed for the protein with and without bound 11-cis-retinaldehyde are associated with protein–ligand interactions. Site directed mutagenesis in conjunction with further NMR and ligand binding studies assures identification of components of the rCRALBP retinoid binding pocket.