Structure, stability and equilibrium (un)folding of flavodoxin

E. Steensma

Research output: Thesisinternal PhD, WU

Abstract

<p>Flavodoxins function as low-potential one-electron carriers using a non-covalently bound FMN cofactor which can exist in three redox states. Flavodoxin structures are characterised by a five-stranded parallel<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" WIDTH="10" HEIGTH="16" ALT="beta" BORDER="0"/>-sheet (order<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>2-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>1-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>3-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>4-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>5) surrounded by<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helices at either side of the sheet. This topology is called the flavodoxin-like fold. In contrast to most folds, the flavodoxin-like fold is shared by many protein superfamilies which are sequentially and evolutionary unrelated.</p><p>Studies on proteins with the flavodoxin-like fold can therefore be utilised to find answers to the so-called protein folding problem which can be captured by the following questions:</p><UL><LI>What is the physical basis of the stability of the folded protein conformation?<LI>What processes or pathways determine which of the many possible conformations is the native folded conformation adopted by the protein?<LI>What are the rules governing the relation between the amino acid sequence and three-dimensional structure of a protein?<LI>Can the three-dimensional structure of a protein be predicted from its amino acid sequence?</UL><p>The research described in this thesis has been carried out to obtain a better understanding of the fundamental rules describing protein folding. This thesis focuses on the structure and stability of <em>Azotobacter vinelandii</em> (strain ATCC 478) flavodoxin II (henceforth designated flavodoxin) in its holo- and apoform (i.e. with and without cofactor FMN, respectively). The results obtained for this particular flavodoxin are compared with those for other flavodoxins, as well as with results obtained for sequentially unrelated proteins having a flavodoxin-like fold. The understanding of the equilibrium (un)folding of flavodoxin is a first step in the characterisation of the energy landscape determining the folding of this protein.</p><p>A general introduction on protein folding, NMR spectroscopy and flavodoxins is presented in Chapter 1.</p><p>To prevent dimerisation during structural and folding studies Cys69Ala and Cys69Ser mutants of wild-type flavodoxin were prepared. pH-Dependent semiquinone/hydroquinone redox potentials of wild-type, Cys69Ala and Cys69Ser flavodoxin were determined using cyclic voltammetry and confirmed by EPR-monitored redox titrations. No significant differences in redox properties of wild-type, Cys69Ala and Cys69Ser flavodoxin are observed. The pH dependence of the semiquinone/ hydroquinone redox potentials can be described using a model assuming two redox-linked protonation sites with a constant redox potential at high pH of -485 ± 4 mV. The electrochemical data which are presented in Chapter 2 show that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue do not alter the dielectric properties and structure of holoflavodoxin.</p><p>In Chapter 3, a new doubly sensitivity-enhanced 3D <sup>1</SUP>H- <sup>15</SUP>N TOCSY-HSQC experiment is described and analysed using the product operator formalism . The overall gain in signal-to-noise ratio obtained using this doubly sensitivity-enhanced TOCSY-HSQC pulse sequence is, compared to the standard (non-enhanced) sequence, 2.49 or 1.89 for spectra obtained for <sup>15</SUP>N-labelled or <sup>15</SUP>N-/ <sup>13</SUP>C-labelled holoflavodoxin samples, respectively. The main factors leading to the signal-to-noise enhancement are the introduction of two enhanced coherence transfer sequences, the elimination of water presaturation and the inclusion of a water flip-back pulse. Incorporation of gradients for coherence pathway selection, however, leads to a reduction in signal intensity.</p><p>The determination of the solution secondary structure of holoflavodoxin is described in Chapter 4. A five-stranded parallel<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-sheet (<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>2-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>1-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>3-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>4-<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>5) is surrounded by five<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helices. The loops extending from the carboxy termini of strands<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>1,<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>3 and<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>4 are involved in FMN binding. Hydrogen/deuterium exchange experiments suggest that (i) amide proton exchange within the core of holoflavodoxin occurs via the apoform of the molecule and that (ii) exchange of the N(3)H proton of FMN only occurs when the cofactor is free in solution. The solvent inaccessibility of the non-polar environment around N(3) could, at least in part, establish the low semiquinone /hydroquinone redox potential. The amide proton exchange rates do not suggest that holoflavodoxin is divided in two subdomains as has been found for the structurally, but not sequentially, homologous protein Che Y. The amide backbone protons of 65 residues and three indole side-chain protons exchange sufficiently slowly (k <sub>ex</sub> &lt; 10 <sup>-5</SUP>s <sup>-1</SUP>) to be able to perform hydrogen exchange pulse labelling experiments and to study the kinetics of flavodoxin folding in great structural detail.</p><p>The structural characteristics of apoflavodoxin as determined by NMR spectroscopy are presented in Chapter 5. Apoflavodoxin has a stable, well-ordered core consisting of a five-stranded parallel<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-sheet surrounded by five<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helices. Large parts of holo- and apoflavodoxin have identical conformations and similar internal dynamics. However, the flavin binding region in apoflavodoxin exhibits considerable conformational dynamics. Flexibility is a likely prerequisite to enable the flavin to enter the interior of the apoprotein. Hydrogen/deuterium exchange measurements suggest that the stable nucleus in apoflavodoxin at least comprises residues in strands<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>1,<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>3,<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>4 and<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>5a and in helices<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>4 and<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>5 in the C-terminal part of the protein. We propose that this is a general feature of flavodoxins. In contrast, the stable nucleus of the sequentially unrelated proteins cutinase and Che Y which share the flavodoxin-like fold is not found in their respective C-terminal parts. The amide proton exchange results show that the stable nucleus may be found in different parts of the flavodoxin-like topology. If folding of flavodoxin is initiated with the collapse of the stable nucleus, as has been found for several other proteins, the folding pathways of structurally homologous proteins seem to be unrelated as well.</p><p>Chapter 6 reflects on the research described in this thesis and combines the NMR studies, as described in Chapters 4 and 5, with equilibrium (un)folding studies on apo- and holoflavodoxin using fluorescence and circular dischroism spectroscopy which have been performed by van Mierlo et al. The following picture for equilibrium (un)folding of flavodoxin arises: (i) holoflavodoxin has a compact stable fold consisting of a five-stranded parallel<img src="/wda/abstracts/i2427_2.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="beta" border="0"/>-sheet surrounded by five<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>-helices; (ii) upon release of the FMN cofactor, apoflavodoxin is formed which has a stable core but a flexible FMN binding region; (iii) in the (un)folding pathway a relatively stable apoflavodoxin folding intermediate is found which is characterised by the loss of tertiary interactions without the complete loss of secondary structure; (iv) the unfolded state of flavodoxin presumably contains some residual structure of an aromatic cluster as a remnant of helix<img src="/wda/abstracts/i2427_1.gif" ALIGN="AbsMiddle" width="10" height="16" ALT="alpha" border="0"/>4. The results obtained on the equilibrium (un)folding of flavodoxin are discussed with respect to the implications for the kinetics of flavodoxin folding.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Laane, N.C.M., Promotor, External person
  • Hilbers, C.W., Promotor, External person
  • van Mierlo, Carlo, Promotor
Award date8 May 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054858300
Publication statusPublished - 1998

Keywords

  • proteins
  • structure

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