TY - JOUR
T1 - What causes hyperfluorescence
T2 - Folding intermediates or conformationally flexible native states?
AU - Ervin, John
AU - Larios, Edgar
AU - Osváth, Szabolcs
AU - Schulten, Klaus
AU - Gruebele, Martin
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health R01 GM057175 (to M.G.), from the National Institutes of Health P41 RR05969-04 (to K.S.), as well as by a computer time grant of the National Science Foundation RCA 935028 (to K.S.). Edgar Larios was recipient of a Beckman Institute Graduate Fellowship while this work was carried out. The authors thank the UIUC Theoretical Biophysics Group where simulations were carried out, and the National Institutes of Health-supported UIUC Laboratory for Fluorescence Dynamics where the anisotropy measurements were made.
PY - 2002
Y1 - 2002
N2 - Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
AB - Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
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U2 - 10.1016/S0006-3495(02)75183-7
DO - 10.1016/S0006-3495(02)75183-7
M3 - Article
C2 - 12080134
AN - SCOPUS:0036286323
VL - 83
SP - 473
EP - 483
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 1
ER -