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Sensitivity of superfolder GFP to ionic agents.
Olesya V Stepanenko1, Olga V Stepanenko1, Irina M Kuznetsova2
1Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
Plos One
|October 28, 2014
Summary
Ionic salts like guanidine thiocyanate alter superfolder green fluorescent protein (sfGFP) spectral features by affecting its chromophore, not just its structure. This impacts fluorescence intensity, a crucial factor for protein folding studies.
Area of Science:
- Biophysics
- Protein Chemistry
- Spectroscopy
Background:
- Superfolder green fluorescent protein (sfGFP) is a valuable tool for studying protein unfolding and refolding due to its stable beta-barrel structure and reversible unfolding.
- sfGFP's ability to fold correctly even in fusion constructs with poorly folded proteins makes it advantageous for various biochemical applications.
- Ionic denaturants are commonly used to investigate protein folding dynamics, but their direct effects on the fluorescent protein's chromophore are not fully understood.
Purpose of the Study:
- To investigate the influence of ionic denaturants and salts, including guanidine thiocyanate, guanidine hydrochloride, sodium chloride, and sodium thiocyanate, on the spectral characteristics of sfGFP.
- To determine whether these agents affect sfGFP structure or its chromophore directly.
- To elucidate the mechanism behind spectral alterations and their implications for fluorescence-based protein studies.
Main Methods:
- Spectroscopic analysis, including absorption and circular dichroism (CD) spectroscopy, was employed to monitor changes in sfGFP.
- sfGFP was exposed to varying concentrations of ionic denaturants and salts.
- Spectral features in the visible range were measured to detect alterations in sfGFP's properties.
Main Results:
- Moderate concentrations of ionic denaturants and salts did not disrupt the overall structure of sfGFP.
- Pronounced alterations in sfGFP's spectral characteristics were observed, indicating specific interactions with the chromophore.
- Changes in absorption and CD spectra suggest specific binding of thiocyanate (SCN-) and chloride (Cl-) anions near the sfGFP chromophore, leading to a redistribution of neutral and anionic chromophore forms and hindering proton transfer, thus decreasing fluorescence intensity.
Conclusions:
- Ionic denaturants and salts can directly affect the chromophore of sfGFP, altering its spectral properties without necessarily unfolding the protein.
- The observed spectral changes are attributed to anion binding in the vicinity of the chromophore, influencing its charge state and proton transfer dynamics.
- Researchers using ionic denaturants in fluorescent protein folding studies must consider the impact on the fluorophore's charge state to accurately interpret results.
