Circular dichroism temperature effects

While it is tempting to explain regulatory and cosolvent effects on the basis of conformational changes favorable or unfavorable to enzyme activity, it is much more difficult to demonstrate the actual involvement, amount, and structural details of such changes. Experimental evidence consists in most cases of bits and pieces provided by techniques such as absorption and fluorescence spectroscopy, circular dichroism, and magnetic circular dichroism. These tools work in solution (and, when desired, at subzero temperatures) to investigate not simply empty enzymes but enzyme—substrate intermediates. However, even with this information, the conformational basis of enzyme activity remains more postulated than demonstrated at the ball and stick level, and in spite of data about the number and sequence of intermediates, definition of their approximate nature, rate constants, and identification of the types of catalysis involved, full explanation of any particular reaction cannot be given and rests on speculative hypothesis. 

These data have led to the development of a catalytic mechanism, shown in Scheme 6, that has been further refined by kinetic isotope effect (KIE) experiments. Substrate binds to Cu(II), replacing bound solvent. The metal coordination facilitates the deprotonation of the substrate hydroxyl group. The proton is transferred to Tyr495, which dissociates from copper. The temperature and pH dependence of the visible absorption and circular dichroism spectra indicate that galactose oxidase exists as an equilibrium of the Tyr495-Cu(II) form (TyroN) and the protonated Tyr495 state. 

The colour of ICPs can also be sensitive to temperature. Substituted polythiophenes, in particular, show a marked blue shift of the highest wavelength absorption band when films or solutions are heated [7-9]. These reversible colour changes have been attributed to a twisting of the polymer backbone to a less ordered non-planar conformation. Less dramatic thermochromic effects have also been reported for polyanilines [3,10,11] where circular dichroism studies of... 

The laser temperature jump instrument can effectively be used to initiate and observe the fast events in protein/peptide folding and unfolding as well as those events that extend out to several milliseconds. In the present study, the unfolding of a helical peptide was determined to occur within tens of nanoseconds, supporting the need for nanosecond or faster initiation techniques. Promising results obtained by the laser temperature jump method will continue to stimulate the development of additional monitoring techniques such as UV absorption and circular dichroism. 

Fig. 7. Temperature effect on the circular dichroism of adenosine-5 -mononicotinate. As the temperature is decreased, more stacking is present. A reciprocal increase in negative ellipticity of the long-wavelength band and an increase in positive ellipticity of the first positive band are observed. From Miles and Urry. ...

The model system that was used to demonstrate the reciprocal relations was adenosine-5 -mononicotinate, which is given in the stacked or interacted conformation in Fig. 6. The temperature effect for this molecule as seen in the circular dichroism spectra is given in Fig. 7, where the reciprocal relations are beautifully apparent. This approach has been used to identify stacked conformations for flavin-adenine dinucleotide and for the oxidized and reduced forms of a- and /3-nicotinamide-adenine dinucleotide. ... 

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