High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms

Photonic activation of plasminogen induced by low dose UVB.

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots

Shortest spatial distances between disulphide bonds and aromatic residues (tryptophan and tyrosine) in full-length native human plasminogen.

<p>The shortest distances (< 6 Å) between atoms of each pair of elements (Trp, Tyr and disulphide bonds) were considered. For Trp and Tyr residues, only the atoms belonging to the indole and benzene rings were considered.</p><p>Shortest spatial distances between disulphide bonds and aromatic residues (tryptophan and tyrosine) in full-length native human plasminogen.</p

Increase in the concentration of detected free thiol groups (open circles) in human plasminogen UV-illumination.

<p>Detection of free thiol groups was carried out using the Ellman’s assay after 280 nm light continuous illumination (22.5 min, 45 min, 90 min, or 112.5 min) of human plasminogen in solution. The experimental values were fitted using an exponential function <i>y = y₀ − A.e^-R0t</i> (fitted curve in red), where <i>y</i> is the concentration of thiol groups (µM) at the 280 nm illumination time <i>t</i> (h), <i>y₀</i> and <i>A</i> are constants and <i>R0</i> is the rate of thiol group formation (h^-1). Fitted experimental parameters were: <i>y₀</i> = 2.29 ± 0.07 µM, <i>A</i> = 3.78 ± 0.42 µM, <i>R0</i> = 0.037 ± 0.005 min^-1. Root mean square error was 99.39%.</p

Shortest spatial distances between disulphide bonds and aromatic residues (tryptophan and tyrosine) in the serine protease domain of human plasminogen.

<p>The shortest distances (<12 Å) between atoms of each pair of elements (Trp, Tyr and disulphide bonds) were considered. For Trp and Tyr residues, only the atoms belonging to the indole and benzene rings were considered.</p><p>* -RH Staple disulphide bonds.</p><p>In bold: disulphide bonds relevant for plasminogen activation.</p><p>Shortest spatial distances between disulphide bonds and aromatic residues (tryptophan and tyrosine) in the serine protease domain of human plasminogen.</p

Fluorescence signal presented by the digested substrate for plasminogen kept in the dark (10 min in the dark, negative control) and UV illuminated plasminogen (10 min 280 nm illumination).

<p>Fluorescence signal is used as a measure of degradation of the substrate and to acess plasmin activity. The fluorescence readings were performed after 1 hour and 22 hours which is standard procedure for the used Casein-based substrate. The background fluorescence signal from substrate blank was subtracted.</p