Authors: Helena Kovacs Alvar Gossert
Publish Date: 2014/01/04
Volume: 58, Issue: 2, Pages: 101-112
Abstract
Three improved 13Cspinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes The first application the HCaliCaroHTOCSY is an HCCHTOCSY in which all CHn moieties of a protein are detected in a single experiment including the aromatic ones This enables unambiguous assignment of aromatic and aliphatic amino acids in a single highly sensitive experiment In the second application the 13Cdetected CallTOCSY magnetization transfer comprises all carbons—aliphatic aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only Thirdly the frequently used HCCCONH experiment was redesigned by replacing the long Ccarbonyl refocused INEPT transfer step by direct 13C–13CTOCSY magnetization transfer from side chain carbons to the backbone carbonyls The resulting HCCCONH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDaWe would like to thank Dr Ēriks Kupče at Bruker BioSpin UK for his kind help in creating an appropriate adiabatic mixing pulse and Dr Wolfgang Bermel Bruker BioSpin Germany for perfecting the pulse sequences We thank Julia Klopp and Aurélie Winterhalter for production of the MBP sample
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