3D J-resolved NMR spectroscopy for unstructured polypeptides: fast measurement of 3J HNH alpha coupling constants with outstanding spectral resolution.
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 (1)H-(15)N SOFAST-HMQC to provide a 3D spectrum with an E.COSY pattern originating from splittings due to (3)J(HNH alpha) and (2)J(NH alpha) couplings. Thereby an effectively homodecoupled (1)H-(15)N correlation spectrum is obtained with significantly improved resolution and greatly reduced spectral overlap compared to standard HSQC and HMQC experiments. The (3)J(HNH alpha) is revealed in three independent ways directly from the peak positions, allowing for internal consistency testing. In addition, the natural H(N) 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 alpha-synuclein.
Lendel
Christofer
C
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Damberg
Peter
P