Phân tích cấu hình bằng các phép đo NOE định lượng của các cặp β-proton qua từng liên kết disulfide trong protein

Journal of Biomolecular NMR - Tập 52 - Trang 127-139 - 2011
Mitsuhiro Takeda1, Tsutomu Terauchi2, Masatsune Kainosho1,2
1Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya, Japan
2Center for Priority Areas, Graduate School of Science and Technology, Tokyo Metropolitan University, Hachioji, Japan

Tóm tắt

Các NOE giữa các β-proton của các dư lượng cysteine qua các liên kết disulfide trong protein cung cấp thông tin trực tiếp về các kết nối và cấu hình của những liên kết chéo quan trọng này, mà những thông tin này thường khó điều tra. Tuy nhiên, với các protein [U-13C, 15N]-thông thường, các quá trình khuếch tán spin nhanh do sự tương tác dipolar mạnh giữa các β-proton geminal cản trở việc đo lường định lượng và do đó cản trở việc phân tích các NOE khoảng cách xa qua các liên kết disulfide. Chúng tôi mô tả một phương pháp chắc chắn để giảm thiểu các khó khăn này, bằng cách sử dụng các protein được gán nhãn chọn lọc với một hỗn hợp cân bằng mol của (2R, 3S)-[β-13C; α,β-2H2] Cys và (2R, 3R)-[β-13C; α,β-2H2] Cys, nhưng hoàn toàn được deuter hóa. Bởi vì một trong hai proton methylene prochiral, tức là β2 (proS) hoặc β3 (proR), luôn luôn được thay thế bằng một deuteron và không proton nào khác còn lại trong các protein được chuẩn bị theo phương pháp gán nhãn này, tất cả bốn NOE dự kiến của các β-proton qua các liên kết disulfide có thể được đo mà không có sự can thiệp khuếch tán spin, ngay cả với thời gian trộn dài. Do đó, các NOE cho các cặp β2 và β3 qua từng liên kết disulfide có thể được quan sát với độ nhạy cao, mặc dù chúng chỉ đạt 25% mức tối đa lý thuyết cho mỗi cặp. Với thông tin NOE, các kết nối liên kết disulfide có thể được xác định một cách rõ ràng cho các protein có nhiều liên kết disulfide. Ngoài ra, các cấu hình xung quanh các liên kết disulfide, cụ thể là χ2 và χ3, có thể được xác định dựa trên khoảng cách proton chính xác của bốn cặp β-proton, thông qua các phép đo định lượng của các NOE qua các liên kết disulfide. Tính khả thi của phương pháp này được chứng minh cho chất ức chế trypsin từ tụy bò, có ba liên kết disulfide.

Từ khóa


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