DETECTING NATIVE-LIKE PROPERTIES IN COMBINATORIAL LIBRARIES OF DE-NOVO PROTEINS

Background: Combinatorial methods based on binary patterning of polar and nonpolar residues have been used to generate large libraries of de novo alpha-helical proteins. Within such libraries, the ability to fi nd structures that resemble natural proteins requires a rapid method t o sort through large collections of proteins and detect those possessi ng 'native-like' features. The current paper presents such a method an d applies it to an initial collection of de novo proteins. Results: We present a method to identify proteins with native-like properties fro m libraries of de novo sequences expressed in vivo. A novel 'rapid pre p' freeze/thaw procedure was used to prepare samples; chromatographic purification was not required. The semi-crude samples were analyzed fo r nativelike features by one-dimensional H-1 NMR spectroscopy. Using t his method, we demonstrate that native-like features can readily be ob served for several proteins among a collection of sequences designed b y binary patterning of polar and nonpolar amino acids. Conclusions: Na tive-like properties can be detected using a method that requires neit her isotopic enrichment nor chromatographic purification. The method i s inexpensive, rapid, and suitable for parallel processing. It can the refore be employed to screen for native-like properties among large co llections of de novo sequences. Using this method, we demonstrate that although the binary code strategy does not explicitly design tertiary packing, it can nonetheless generate proteins that possess native-lik e properties. The use of combinatorial methods to produce large collec tions of proteins coupled with the availability of a rapid assay for d etecting native-like properties will facilitate the design and isolati on of novel proteins with desirable properties.