Irina Pozdnyakova

Approaching the speed limit for Greek Key β-barrel formation: transition-state movement tunes folding rate of zinc-substituted azurin

Azurin is a blue-copper protein with a beta-barrel structure of Greek Key topology. In vitro, copper can be substituted with zinc without change in protein structure. We here analyze the kinetic folding behavior of zinc-substituted Pseudomonas aeruginosa azurin. Our findings can be summarized in three key conclusions: first, zinc remains strongly bound to the polypeptide upon unfolding, suggesting that the cofactor may bind to the protein before polypeptide folding in vivo. Second, the semi-logarithmic plot of folding and unfolding rates for zinc-substituted azurin as a function of denaturant concentration exhibits curvature due to a changing transition-state structure. Third, the extrapolated folding speed in water for zinc-substituted azurin is similar to that of other proteins with the same topology, implying that there is a speed limit that can be modulated by stability-driven transition-state movement for formation of beta-barrel structures with Greek Key topology.

Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin.

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (CuII and CuI) and zinc affinities for folded and unfolded azurin polypeptides, respectively.

Studies of Pseudomonas aeruginosa Azurin Mutants: Cavities in β-Barrel Do Not Affect Refolding Speed

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.

New insights into Fragile X syndrome

Lack of functional Fragile X mental retardation protein (FMRP) is the primary cause of the Fragile‐mental retardation syndrome in humans. In most cases, the disease results from transcriptional silencing of fragile mental retardation gene 1, fmr1, which encodes FMRP. However, a single missense mutation (I304N) in the second KH domain of FMRP gives rise to a particularly severe case of Fragile X syndrome. A Drosophila homolog of FMRP has been identified, Drosophila Fragile X related protein (dFXRP). The corresponding missense mutation in dFXRP, the I307N, has pronounced effects on the in vivo activity of the protein. The effect of the point mutation on the structure and function of FMRP is unclear, and published data are contradictory. No in vitro structural or stability studies have been performed on dFXRP. Here we show that a construct that contains only the tandem KH1‐KH2 domains is a stable, well‐folded unit suitable for detailed structural and functional characterization. Using this KH1‐KH2 construct we explicitly test a hypothesis that has been proposed to explain the effect of the Ile→Asn mutation: that it causes complete unfolding of the protein. Here we show that the I307N point mutation does not completely unfold the KH domain. The KH1‐KH2 construct bearing I307N substitution is stable in isolation and adopts a native‐like fold. Thus our data favor alternative explanations for the in vivo observed loss of dFXRP activity associated with I307N mutation: (a) the point mutation might affect intra and/or inter‐molecular interactions of dFXRP; or (b) it might impair dFXRPs interactions with its RNA target(s).

If space is provided, bulky modification on the rim of Azurin’s β-barrel results in folded protein

Pseudomonas aeruginosa azurin is a blue‐copper protein with a β‐barrel fold. Here we report that, at conditions where thermal unfolding of apo‐azurin is reversible, the reaction occurs in a single step with a transition midpoint (T m) of 69°C (pH 7). The active‐site mutation His117Gly creates a cavity in the β‐barrel near the surface but does not perturb the overall fold (T m of 64°C, pH 7). Oxidation of the active‐site cysteine (Cysteine‐112) in wild‐type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native‐like structure. In sharp contrast, Cysteine‐112 oxidation in His117Gly azurin yields a modified apo‐azurin that appears folded and displays cooperative, reversible unfolding (T m∼55°C, pH 7). We conclude that azurins β‐barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.