Time resolved emissions in the picosecond range of single tryptophan recombinant myoglobins reveal the presence of long range heme protein interactions.
We have analyzed the time resolved fluorescence emission in the subnanosecond range of recombinant wild-type SW myoglobin and its single TRP mutants W7F and W14F. These recombinants carry a methionine at the N1 terminal end. The emission of Trp-7 in the met form of W14F showed residual lifetime components much shorter than those estimated after excitation energy transfer to the heme. We propose that in this recombinant the N1 methionine is close to Trp-7, thereby producing an extra quenching due to either collisions or electron transfer with its sulfur. When the measurements were repeated on its CO-form, the extra quenching of Trp-7 was much decreased, indicating a heme linked conformational change involving the amino terminal end of the protein. This hypothesis is supported by ligand linked conformational changes in myoglobin, reported by Ansari et al. and by Giardina et al. At neutral pH the lifetimes of W7F were consistent with estimations based on the atomic coordinates of SW myoglobin. Those of the wild-type were exactly the combination of the lifetimes of the two mutants. This suggest that the mutations did not affect the overall structure of the protein. However, in the ferric form, substitution of Trp-14 in W14F resulted in low stability at acid pH, as evident from lifetimes modifications at pH 4.8, while no modifications were produced by titrations of W7F to pH 4.5. This suggests a role of Trp-14 in the structural stability of myoglobin.
Gryczynski
Z
Z
Department of Biochemistry, University of Maryland Medical School, Baltimore 21201-1503, USA.
Bucci
E
E
D000818:Animals; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*; D014907:Whales
D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D014364:Tryptophan