| 1. |
- Engman, Cecilia, 1974, et al.
(author)
-
Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin.
- 2004
-
In: Protein Science. ; 13:10, s. 2706-2715
-
Journal article (peer-reviewed)abstract
- The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state.
|
|
| 2. |
- Grünberg, Raik, et al.
(author)
-
Complementarity of structure ensembles in protein-protein binding.
- 2004
-
In: Structure. ; 12:12, s. 2125-36
-
Journal article (peer-reviewed)abstract
- Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.
|
|
| 3. |
- Leckner, Johan, 1969
(author)
-
Folding and Structure of Azurin - The Influence of a Metal
- 2001
-
Doctoral thesis (other academic/artistic)abstract
- The structural role of the metal in the protein azurin from Pseudomonas aeruginosa has been a long standing question. Azurin belongs to the cupredoxin family and is a 128 residue beta-barrel protein of greek-key topology. The biological function of azurin is that of an electron carrier, and the electron shuttling is mediated via a redox active copper ligand that is coordinated by the protein. Another structural feature of azurin is an N-terminal disulfide bond that is not conserved within the cupredoxin family. This thesis contains the near complete NMR assignments, as well as the solution structure of diamagnetic (Cu(I)) azurin. Furthermore, assignments of the contact shifted residues in the paramagnetic (Cu(II)) form and an investigation of the pseudocontact shift contribution is presented. The investigation of the folding covers different azurin species: metal ligand substitutions, engineered apo-mutants and mutants lacking the disulfide bond. Equilibrium folding investigations show that the metal ion remains bound to the protein in the denatured state, and that azurin is more stable in the oxidized Cu(II) form than in the reduced Cu(I) form. The stabilization is primarily an entropic destabilization of the unfolded state, and is ascribed to a tetragonal metal coordination in the oxidized form that changes to trigonal upon reduction. From kinetic folding measurements, the influence of the metal ion and the disulfide bond on the folding process can be deduced. The folding behavior is significantly different in the apo-form, compared to the metal bound form, changing from a two-state to a three-state process. Removing the disulfide bond only affects the folding and unfolding rates. It is found that neither the metal, nor the disulfide bond, are important for the folding of azurin, but that both significantly stabilize azurin by reducing the unfolding rate.
|
|
| 4. |
- Sandberg, Anders, 1975, et al.
(author)
-
Apo-azurin folds via an intermediate that resembles the molten-globule.
- 2004
-
In: Protein Science. ; 13:10, s. 2628-2638
-
Journal article (peer-reviewed)abstract
- The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.
|
|
