Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement
| Autor: | Carlo P. M. van Mierlo, Adrie H. Westphal, Bregje J. de Kort, Sanne M. Nabuurs |
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| Rok vydání: | 2010 |
| Předmět: |
Models
Molecular Protein Denaturation Flavodoxin Membrane transport and intracellular motility [NCMLS 5] Plasma protein binding folding mechanism Biochemistry Protein Structure Secondary nmr molten globule chemistry.chemical_compound α–β Parallel protein hydrogen-exchange CMTSL flavodoxin-ii Protein secondary structure Renal disorder [IGMD 9] Mesylates biology Temperature General Medicine Paramagnetic relaxation enhancement Molten globule Folding (chemistry) Protein folding Hydrophobic and Hydrophilic Interactions Protein Binding MTSL Biophysics Biochemie beta parallel protein Hydrophobic effect Cyclic N-Oxides Magnetics Cysteine denatured state Guanidine VLAG Azotobacter vinelandii Original Paper Dose-Response Relationship Drug pathway Crystallography chemistry Amino Acid Substitution Unfolded protein native-like biology.protein Spin Labels azotobacter-vinelandii apoflavodoxin Apoproteins |
| Zdroj: | European Biophysics Journal European Biophysics Journal with Biophysics Letters, 39, 4, pp. 689-98 European Biophysics Journal, 39(4), 689-698 European Biophysics Journal 39 (2010) 4 European Biophysics Journal with Biophysics Letters, 39, 689-98 |
| ISSN: | 0175-7571 |
| Popis: | Item does not contain fulltext 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 alpha-beta 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 alpha-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. 01 maart 2010 |
| Databáze: | OpenAIRE |
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