Abstract

Nuclear magnetic resonance spectroscopic structure determination of proteins has been under rapid development during the last decade. The size limitation impeding structural studies of biological macromolecules in solution has increased from 10 kDa to 30 kDa thanks to exploitation of ¹⁵N/¹³C enrichment. Perdeuteration of non-exchangeable protons has pushed this limit even further, allowing backbone resonance assignment of 40 to 50 kDa proteins. Most recently, transverse relaxation optimized spectroscopy (TROSY) has been demonstrated to lengthen ¹⁵N and ¹H^N spin transverse relaxation times significantly, especially in large perdeuterated proteins, thus extending the size limit beyond 100 kDa systems. However, determination of structurally important nuclear Overhauser enhancements (NOE) suffers from perdeuteration, due to the lower density of proton spins available, eventually leading to imprecise protein structures. Very recently, residual dipolar couplings have been used to supplement NOE information, enabling accurate molecular structures to also be obtained with perdeuterated proteins. This thesis focuses on the measurement of the structurally important ³J-coupling between ¹H^N and ¹H^α spins, and determination of residual dipolar couplings by utilizing the novel spin-state-selective subspectral editing together with the TROSY methodology. This approach allows precise measurement of a large number of ipolar couplings in larger protonated or perdeuterated proteins.