3D J-resolved NMR spectroscopy for unstructured polypeptides: fast measurement of 3J_HNHα coupling constants with outstanding spectral resolution

摘要

A powerful experiment for the investigation of conformational properties of unstructured states of proteins is presented. The method combines a phase sensitive J-resolved experiment with a 1H-15N SOFAST-HMQC to provide a 3D spectrum with an E.COSY pattern originating from splittings due to 3J_HNHα and 2J_NHα couplings. Thereby an effectively homodecoupled 1H-15N correlation spectrum is obtained with significantly improved resolution and greatly reduced spectral overlap compared to standard HSQC and HMQC experiments. The 3J_HNHα is revealed in three independent ways directly from the peak positions, allowing for internal consistency testing. In addition, the natural HN linewidths can easily be extracted from the lineshapes. Thanks to the SOFAST principle, the limited sweep width needed in the J-dimension and the short phase cycle, data accumulation is rapid with excellent sensitivity per time unit. The experiment is demonstrated for the intrinsically unstructured 14 kDa protein α-synuclein.