Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

• Published in: Journal of biomolecular NMR Vol. 16; no. 3; pp. 209 - 219
• Main Authors: McDermott, A, Polenova, T, Bockmann, A, Zilm, K W, Paulson, E K, Martin, R W, Montelione, G T, Paulsen, E K
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01.03.2000
• Subjects: Amino acids; Aprotinin - chemistry; Binding sites; Carbon Isotopes; Models, Molecular; Nitrogen Isotopes; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1023/A:1008391625633

We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-13C,15N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical 13C peak line widths are 0.5 ppm, resulting in Calpha-Cbeta and Calpha-CO regions that exhibit many resolved peaks. Two-dimensional carbon-carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 micromol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.