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Ultrafast, intra‐oligomer fluorescence resonance energy transfer (FRET) between an immature green‐emitting GFP‐like chromophore to the mature red‐emitting chromophore is found in the novel red fluorescent protein wt‐DsRed (the picture shows the steady‐state absorption (solid line) and emission (dotted) spectra). Since FRET is by its very nature a short range process, it represents a highly suitable method to probe oligomerization. This work describes a method preferentially applicable to the efficient screening of protein variants with mutagenetically altered surface docking sites.

The mechanism of photoinduced hole injection into DNA has been studied using an integrated approach that combines NMR structural analysis, time-resolved spectroscopy, and quantum-chemical calculations. A covalently linked acridinium derivative, the protonated 9-amino-6-chloro-2-methoxyacridine (X+), is replacing a thymine and separated from either guanine (G) or the easier to oxidize 7-deazaguanine (Z) by one adenine.thymine (A.T) base pair. The key features of this donor/acceptor system are the following: (i) In more than 95% of the duplexes, X+ is located in a central, coplanar position between the neighboring A.T base pairs with its long axis in parallel showing minimal twist and tilt angles (<15 degrees). The complementary adenine base is turned out into the extrahelical space. In a minority of less than 5%, X+ is found to be still attached to the duplex. X+ is most probably associated with one of the phosphates, since it is neither intercalated between more remote base pairs nor bound to sugars or grooves. This minority characterized by an excited state lifetime >10 ns gives rise to a small background signal in time-resolved measurements and contributes predominantly to steady-state fluorescence spectra. (ii) Although the intercalation mode of X+ is well defined, the NMR structure reveals that there are two conformations of X+ with respect to the arrangement of its methoxy substituent. In one conformation, the methoxy group is in the plane of the chromophore, while, in the other extraplanar conformation, the methoxy group forms an angle of 70 degrees with the acridinium ring. The fluorescence decay of 5'-ZAX and 5'-GAX tracts can be fitted to a biexponential function with similar amplitudes, reflecting the oxidation dynamics of G and Z, with the slower rate being determined by larger thermal activation energy. The attribution of biexponential electron transfer (ET) dynamics to the bimodal orientation of the methoxy group at the acridinium is supported by quantum-chemical calculations. These predict a larger free energy change for hole transfer in the nonplanar conformation as compared to the planar one, whereas the difference in the electronic couplings is negligible. (iii) Kinetic studies of the directionality of the 1(X+)* induced hole injection reveal similarly fast decay components in both directions of the duplex, that is, in 5'-ZAX and 5'-XAZ, with the amplitude of the fast component being significantly reduced in 5'-XAZ. The NMR structure shows that local structural deviations from B-DNA are much more pronounced in the 3'-5' direction than in the 5'-3' direction. According to quantum-chemical calculations, the directionality of charge injection is not a universal feature of the DNA duplex but depends critically on the rotation angle of the aromatic plane of the acridinium within the pi stack. The arrangement of X+ in 5'-ZAX and 5'-XAZ corresponds to a conformation with weak directionality of the electronic couplings. The increased disorder in the 3'-5'direction favors slow hole transfer components at ...

Ultrafast, intra‐oligomer fluorescence resonance energy transfer (FRET) between an immature green‐emitting GFP‐like chromophore to the mature red‐emitting chromophore is found in the novel red fluorescent protein wt‐DsRed (the picture shows the steady‐state absorption (solid line) and emission (dotted) spectra). Since FRET is by its very nature a short range process, it represents a highly suitable method to probe oligomerization. This work describes a method preferentially applicable to the efficient screening of protein variants with mutagenetically altered surface docking sites.