Chromophore modification

The steps leading to the formation of the intrinsic chro-mophore have recently been investigated kinetically with S65T-GFP. The process of chromophore formation is an ordered sequence of three distinct steps (1) slow protein folding (kf = 2.44 X 10 s ) that precedes chromophore modification (2) an intermediate step occurs that includes, but may not be necessarily limited to, cycli-zation of the tripeptide chromophore motif (kc = 3.8 X 10 s ) and (3) rate-limiting oxidation of the cyclized chromophore (kox = 1 51 X s ). Reid and Flynn also reasoned that because chromophore forms de novo from purified denatured protein and is a first-order process, GFP chromophore formation is likely to be an autocatalytic process. 

Gamma-Ray Induced Chromophore Modification of Softwood Thermomechanical Pulp... 

It is important to point out that this synthetic route has been optimized for the incorporation of ethidium as an artificial DNA base and therefore cannot simply be transferred for the preparation of other chromophores as DNA base surrogates. This makes it clear that this type of DNA-chromophore modification represents the most time-consuming one and much synthetic research effort needs to be invested in order to obtain a reliable synthetic procedure for the routine synthesis of chromophore-modified DNA. 

Another type of chromophore modification can be accomplished by extension of the unsaturated chain length. The spectral responses of poly(vinyl acetate cinnamate) and poly(vinyl acetate cinnamylidene acetate) (Fig. 6) typify this method 39>. 

Chromophore modifications 1) Reduction with borohydride (modified from ref. 10). A solution of PC or isolated subunits (chromophore concentration 7-21/iM, 0.9M potassium phosphate, pH 7, 8M urea) was treated with NaBH (170 mM). After complete reduction (spectrum, 45 min) excess reductant was destroyed by glucose. For reoxidation experiments, the samples (lOOmM potassitim phosphate, pH 7, containing 70% ammonium sulfate to prevent protein degradation) were allowed to stand at room temperature in the dark for up to nine days. This was followed by recombination (if not yet done), denaturation (8M urea in lOOmM potassium phosphate, pH7) and renaturation as described below. 2) Photobleaching of the o-subunit (8/xM protein, 100 mM phosphate, 8M urea, pH 7.5) was... 

Indirect chromophore modification was done by titration of trimeric phycocyanin (oB) with PCMS (para-chloro-mercury-benzenesulfonate) in a 1.1-1.2 fold excess (12). 

To date, we have attempted only minor chromophore modifications which can be effected in a cost effective manner necessary to produce the quantities of materials (grams to kilograms) necessary for prototype device studies (Figure 4). 

Fig. 2 Sdiematk of folding, chromophore formation, and chromophore modification of DsRed. The p-can structure represents the native conformation of the protein, while the denatured form is shown as an irregular chain. It is not known at which step oligomerization occurs, n-con-jugation for visible-light absorption is indicated in gray. Reprinted with permission from Miyawaki et al., Curr. Opin. Chem. Biol. 7,560 (2003). Copyright 2003 Elsevier...

The test is based on chromophore modification resulting from the transformation of a 5,6-epoxide group to 5,8-epoxide in acid medium. This stmctural transformation means the loss of the conjugated double bond at positions 7 and 8 of the central polyene, causing a hypsochromic displacement of the absorption spectmm of 15-20 nm. Chromatographically, the 5,8-epoxide derivatives are more polar, so that chromatographic development by TLC using silica gel as support reveals bands with lower Rf values. 

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