UPLABEL

Optimization of amino acid type- specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional methods. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i – 1) and 15NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with 13C and the second with 15N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of ¬ unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B2R).