Original Paper

European Biophysics Journal

, Volume 39, Issue 4, pp 689-698

First online:

Open Access This content is freely available online to anyone, anywhere at any time.

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

  • Sanne M. NabuursAffiliated withLaboratory of Biochemistry, Wageningen UniversityDepartment of Physiology, Radboud University Nijmegen Medical Center
  • , Bregje J. de KortAffiliated withLaboratory of Biochemistry, Wageningen UniversityDepartment of Biomedical Analysis, Utrecht University
  • , Adrie H. WestphalAffiliated withLaboratory of Biochemistry, Wageningen University
  • , Carlo P. M. van MierloAffiliated withLaboratory of Biochemistry, Wageningen University Email author 


Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.


Unfolded protein Flavodoxin α–β Parallel protein Paramagnetic relaxation enhancement MTSL CMTSL