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The structure and dynamics of partially folded actin
K K Turoverov1, A G Biktashev, S Y Khaitlina
1Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia. kkt@mail.cytspb.rssi.ru
Biochemistry
|May 13, 1999
Summary
Inactivated actin exhibits exposed hydrophobic clusters and restricted tryptophan residue movement, suggesting it may be an intermediate in protein folding. This state is stable across various conditions and refolding pathways.
Area of Science:
- Biochemistry
- Protein structure and dynamics
Background:
- Actin is a crucial protein involved in muscle contraction and cellular structure.
- Understanding actin's structural transitions, particularly inactivation, is vital for comprehending its biological functions and potential dysfunction.
Purpose of the Study:
- To investigate the structural changes in inactivated alpha-actin.
- To determine the nature of exposed hydrophobic regions and tryptophan residue environments in inactivated actin.
- To explore the potential of inactivated actin as an intermediate in the protein folding-unfolding pathway.
Main Methods:
- Steady-state and time-resolved intrinsic fluorescence spectroscopy.
- Fluorescence quenching assays using acrylamide.
- Surface hydrophobicity assessment with 8-anilino-1-naphthalenesulfonic acid (ANS).
- Sedimentation analysis, fluorescence anisotropy, and circular dichroism (CD) spectroscopy.
Main Results:
- Inactivated actin shows significantly increased ANS binding, indicating solvent-exposed hydrophobic clusters.
- Actin macromolecules associate specifically, with sedimentation constants shifting from 3 S (native) to 20 S (inactivated), leading to irreversible transitions.
- Despite a red-shifted fluorescence spectrum, inactivated actin exhibits reduced acrylamide quenching efficiency, suggesting tryptophan residues are shielded within a packed polar environment.
- Restricted oscillations of tryptophan residues were observed in inactivated actin compared to native actin.
Conclusions:
- Inactivated actin possesses a unique structure with exposed hydrophobic clusters and a well-defined internal environment for tryptophan residues.
- The observed structural properties are invariant to experimental conditions, inactivation methods, and protein concentration.
- Inactivated actin demonstrates thermodynamic stability and secondary structure, supporting its role as a potential intermediate in the actin folding-unfolding pathway.