Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.
We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin alpha suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.
Nettels
Daniel
D
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Müller-Späth
Sonja
S
Küster
Frank
F
Hofmann
Hagen
H
Haenni
Dominik
D
Rüegger
Stefan
S
Reymond
Luc
L
Hoffmann
Armin
A
Kubelka
Jan
J
Heinz
Benjamin
B
Gast
Klaus
K
Best
Robert B
RB
Schuler
Benjamin
B
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019791:Guanidine / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis; D013696:Temperature; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D006360:Heat-Shock Proteins; D011498:Protein Precursors; C040782:prothymosin alpha; D013947:Thymosin; D019791:Guanidine