Simultaneous processing of solid-state NMR relaxation and 1D-MAS exchange data: the backbone dynamics of free vs. binase-bound barstar Alexey G. Krushelnitsky a, * , Gu ¨nter Hempel b , Detlef Reichert b a Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, P.O. Box 30, 420111, Russian Federation b Physics Department, Halle University, Friedemann-Bach-Platz 6, Halle 06108, Germany Received 20 February 2003; received in revised form 4 June 2003; accepted 19 June 2003 Abstract Two types of dynamic solid-state NMR experiments—relaxation and 1D-MAS exchange—were combined for the investigation of the backbone dynamics of a 15% randomly 15 N-enriched protein barstar in both free and binase-bound states. The main novelty of this work is a simultaneous quantitative processing of the results of these two types of experiments that we call Simultaneous Relaxation and Exchange Data Analysis (SREDA) approach. It extends the well-known model-free approach such that it permits to discriminate between various motional models (jumps between different sites, wobbling in a cone, etc.). This objective cannot be achieved by analyzing the relaxation or exchange data separately. The SREDA approach was applied to probe a modification of the average backbone dynamics of barstar upon forming a complex with another protein binase. T 1 and off-resonance T 1q relaxation times of 15 N backbone nuclei were measured at three temperatures between 0 and 45 jC, 1D-MAS exchange (CODEX) data were obtained at room temperature within the mixing time range from 0.3 to 200 ms. It has been found that the barstar backbone participates in two molecular processes with correlation times in the 10  9 –10  7 and 10  3 –10  2 s ranges. Forming the complex with binase results in a significant decrease of the amplitudes of both motions, suggesting that the complex is a more rigid and stable structure than free barstar. D 2003 Elsevier B.V. All rights reserved. Keywords: NMR; Relaxation; Exchange; CODEX; Dynamic; Correlation function 1. Introduction During the last few years, NMR studies of protein dynamics in solid-state have acquired growing attention. In addition to the obvious fact that membrane proteins can be studied only in their native insoluble state, solid-state NMR appears to be an indispensable tool for studying molecular dynamics of soluble proteins as well. Since Brownian overall tumbling does not occur in solids and thus does not overlap with all slower internal motions, the study of the latter becomes accessible by a greater number of NMR techniques than in solution. From the viewpoint of understanding mechanisms of protein function, these slow motions are very likely more important than motions in the sub-nanosecond range that are being studied in detail by liquid-state NMR because the former occur on the same time scale as many biologically relevant events, like folding, binding, catalysis, etc. Until the end of the 1990s there were, in practice, only two NMR methods available that could be applied to solid- state protein dynamics studies—line shape analysis of static or slowly rotating samples and relaxation [1]. Recently, theoretical and experimental advances in solid-state magic angle spinning (MAS) exchange techniques have made them applicable to complex systems like proteins. The main methodological achievement was the reduction of the di- mensionality of the originally proposed 2D-MAS exchange methods [2] to 1D-methods, without loss of dynamic information. The corresponding gain in machine time and signal-to-noise ratio paved the way for many applications. The 1D-MAS exchange methods, time-reversed ODESSA [3] and CODEX [4], have already been employed for investigation of several proteins [5–8] and shown to be powerful tools for studying molecular dynamics in the millisecond range of correlation times. In the present work, we apply Simultaneous Relaxation and Exchange Data Analysis (SREDA) for the study of 1570-9639/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1570-9639(03)00207-3 * Corresponding author. Tel.: +7-8432-387-266; fax: +7-8432-387- 577. E-mail address: Krushelnitsky@mail.knc.ru (A.G. Krushelnitsky). www.bba-direct.com Biochimica et Biophysica Acta 1650 (2003) 117 – 127