Crystalline ribonuclease A loses function below the dynamical transition at 220 K

Area of Science:

• Biophysics
• Structural Biology
• Enzymology

Background:

• Proteins exhibit biphasic dynamic behavior across temperatures, with a notable transition around 220 K.
• This transition is observable through various biophysical techniques including X-ray crystallography, Mössbauer scattering, and neutron scattering.

Purpose of the Study:

• To investigate the role of protein flexibility in enzyme function.
• To determine the temperature-dependent binding properties of ribonuclease A (RNase A).

Main Methods:

• High-resolution X-ray diffraction was used to study crystalline ribonuclease A.
• Binding assays were performed at temperatures below (212 K) and above (228 K) the observed dynamic transition.
• Molecular dynamics simulations were also employed to understand atomic motion at different temperatures.

Main Results:

• Ribonuclease A (RNase A) failed to bind substrate or inhibitor at 212 K.
• Rapid substrate and inhibitor binding occurred at 228 K.
• Once bound at the higher temperature, the inhibitor remained bound even after cooling the enzyme below the transition temperature.

Conclusions:

• Enzyme flexibility, particularly above the 220 K transition, is essential for substrate and inhibitor binding.
• These findings suggest a direct link between protein dynamics and catalytic activity.
• The temperature-dependent binding indicates a conformational change required for enzyme function.