The Function of a Protein is Intrinsically Linked to the Identity of its Active-Site and Ligand
FAST-NMR combines structural biology and NMR ligand affinity screens with bioinformatics to assign a function to a hypothetical protein or a protein of unknown function. This is based on basic tenets of biochemisttry where proteins with similar functions will have interactions are determined through a tierd NMR screen using a library composed of compounds with known biological activity. A rapid protein-ligand co-structure is determined by combining the experimental identification of the ligand-binding site from NMR chemical shift perturbations with the protein-ligand docking program AutoDock. Our CPASS (Comparison of Protein Active Site Structures) software and database is then used to compare this active site with proteins of known function.
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Our functional chemical library is first screened as mixtures using a 1D NMR line-broadening screen to rapidly identify binders. The relative change in peak intensity or line-width is used to measure a semi-quantitative dissociation constant (KD) that allows the ligands to be ranked. Only the ligands that exhibit a positive binding interaction in the 1D NMR line-broadening screen are further screened as singletons by measuring chemical shift changes in 2D 1H-15N HSQC spectra.
The observation of a consensus clustering of residues that experienced a chemical shift perturbation (CSP) on the molecular surface of the protein confirms a specific, stoichiometric binding interation for the ligand and the ligand-defined binding site or the proteins active-site/functional-site.
The CSPs are used in combination with AutoDock to rapidly determine (~30-45 min.) a protein-ligand co-strucure. The co-structure is then used as input for CPASS to identify potential functional homologs based on the sequence and structure similarity of ligand-defined binding sites (higher information content than global sequence or structure similarity searches).
Approximately 50% of the genome of multiple organisms contain proteins of unknown function, where 30-50% of these proteins are amenable to NMR analysis.
In cases where a structure and/or NMR assignments of a hypothetical protein is unavailable, FAST-NMR can provide functional information by the similarity in the ligand-binding profile. Comparable to sequence homology, the relative KD measured for each ligand against a protein of unknown function can be compared against this same list of binding affinities for proteins of known function. A similarity score can be obtained based on the relative differences in the individual KD values. A function can then be implied based on a high similarity in the ligand binding profiles.
Also, the output of FAST-NMR is a protein-ligand co-structure, which may provide the starting point for a structure-based drug design effort. This is especially true since the compounds in the functional chemical library have "drug-like" characteristics.
References
- (58) K. A. Mercier, M. Baran, V. Ramanathan, P. Revesz, R. Xiao, G. T. Montelione and R. Powers* (2006) "FAST-NMR - Functional Annotation Screening Technology Using NMR", J. Amer. Chem. Soc., 128(47):15292-15299. PMC2529462.
- (62) R. Powers*, J. Copeland and K. Mercier (2008) "The Application of FAST-NMR for the Identification of Novel Drug Discovery Targets", Drug Discov. Today, 13(3-4):172-179. PMC18275915.
- (61) R. Powers* (2007) "Functional Genomics and NMR Spectroscopy", Comb. Chem. High Throughput Screening, 10(8):676-697. PMID18045080.
Picture Gallery
- From: JACS (2006)
- Process Flow Chart of FAST-NMR
- Functional assignments for hypothetical protein SAV1430 from S. aureus
- Sequence alignment of SAV1430 with NifU
- Hypothetical Proteins from Structural Genomics
- From: Drug Discovery Today (2008)