TY - JOUR
T1 - Time resolved emissions in the picosecond range of single tryptophan recombinant myoglobins reveal the presence of long range heme protein interactions
AU - Gryczynski, Zygmunt
AU - Bucci, Enrico
N1 - Funding Information:
We are indebted to Eileen S. Singleton for the preparation of the recombinant myoglobins and to Emily E. Scott and John S. Olson for inspecting the manuscript and fruitful discussions. This work was supported in part by grants P01-HL-48517 (EB), R01-GM-35649(JSO) and RR-08119(CFS). Computer time and facilities were supported in part by the computer network of the University of Maryland.
PY - 1998/9/14
Y1 - 1998/9/14
N2 - 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. Copyright (C) 1998 Elsevier Science B.V. All rights reserved.
AB - 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. Copyright (C) 1998 Elsevier Science B.V. All rights reserved.
KW - Lifetimes
KW - Recombinant
KW - SW myoglobin
KW - Single tryptophan
UR - http://www.scopus.com/inward/record.url?scp=0032517044&partnerID=8YFLogxK
U2 - 10.1016/S0301-4622(98)00181-1
DO - 10.1016/S0301-4622(98)00181-1
M3 - Article
C2 - 9779582
AN - SCOPUS:0032517044
SN - 0301-4622
VL - 74
SP - 187
EP - 196
JO - Biophysical Chemistry
JF - Biophysical Chemistry
IS - 3
ER -