[6] Rapid Assessment of Protein Structural Stability and Fold Validation via NMR By Bernd Hoffmann,Christian Eichmu ¨ ller, Othmar Steinhauser, and Robert Konrat Abstract In structural proteomics, it is necessary to efficiently screen in a high- throughput manner for the presence of stable structures in proteins that can be subjected to subsequent structure determination by X-ray or NMR spectroscopy. Here we illustrate that the 1 H chemical distribution in a protein as detected by 1 H NMR spectroscopy can be used to probe protein structural stability (e.g., the presence of stable protein structures) of pro- teins in solution. Based on experimental data obtained on well-structured proteins and proteins that exist in a molten globule state or a partially folded -helical state, a well-defined threshold exists that can be used as a quantitative benchmark for protein structural stability (e.g., foldedness) in solution. Additionally, in this chapter we describe a largely automated strategy for rapid fold validation and structure-based backbone signal assignment. Our methodology is based on a limited number of NMR experiments (e.g., HNCA and 3D NOESY-HSQC) and performs a Monte Carlo–type optimization. The novel feature of the method is the opportu- nity to screen for structural fragments (e.g., template scanning). The per- formance of this new validation tool is demonstrated with applications to a diverse set of proteins. Introduction The genome sequencing projects are delivering vast amounts of protein sequences encoding functionally important proteins, which are putative protein therapeutics and/or targets for the pharmaceutical industry. The concept of ‘‘structural proteomics’’ or ‘‘structural genomics’’ [e.g., the elucidation of the three-dimensional (3D) structures of the encoded pro- teins] is based on the empirical finding that protein function cannot always be deduced from the primary sequence but is coded in its 3D shape (Jones and Thornton, 1997; Kasuya and Thornton, 1999; Russell, 1998; Russel et al., 1998; Thornton et al., 1991). Beyond that, structural proteomics efforts will also enlarge the database of known protein structures and provide a sufficiently large basis set of structures to allow for an efficient 142 proteomics [6] Copyright 2005, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 394 0076-6879/05 $35.00