How to experimentally determine hydrophobic patches on a protein

Experimental determination of hydrophobic patches on a protein typically involves techniques that exploit the hydrophobicity of certain probes or ligands to interact selectively with hydrophobic regions of the protein. Here are some commonly used experimental methods:

1. Hydrophobicity Probes:

   • Certain fluorescent dyes or probes are hydrophobic and preferentially bind to exposed hydrophobic regions of proteins.
   • One example is 8-anilino-1-naphthalenesulfonic acid (ANS), which exhibits enhanced fluorescence upon binding to hydrophobic patches.
   • By monitoring changes in fluorescence intensity or spectral properties of these probes in the presence of the protein, hydrophobic patches can be identified.
   • Fluorescence spectroscopy or fluorescence microscopy can be used to visualize the binding of hydrophobic probes to the protein.

2. Hydrophobic Interaction Chromatography (HIC):

   • In HIC, a hydrophobic stationary phase is used to separate proteins based on their hydrophobicity.
   • Proteins with exposed hydrophobic patches interact more strongly with the stationary phase and elute later, while proteins with fewer hydrophobic patches elute earlier.
   • By analyzing the elution profile of the protein on the HIC column, hydrophobic patches can be inferred.

3. Thermal Shift Assay (TSA):

   • TSA measures the thermal stability of proteins in the presence of various ligands or additives.
   • Hydrophobic ligands or denaturants can bind to exposed hydrophobic patches, stabilizing the protein structure and increasing its thermal stability.
   • By monitoring changes in protein unfolding temperature (Tm) in the presence of different ligands, hydrophobic patches can be identified.

4. Hydrophobicity Partitioning:

   • Hydrophobicity partitioning assays involve partitioning proteins between hydrophilic and hydrophobic phases.
   • Proteins with exposed hydrophobic patches preferentially partition into the hydrophobic phase.
   • By quantifying the distribution of the protein between the phases, the hydrophobicity of the protein and the location of hydrophobic patches can be determined.

5. Limited Proteolysis:

   • Limited proteolysis involves digesting the protein with a protease under controlled conditions.
   • Proteases preferentially cleave exposed or flexible regions of the protein, including hydrophobic patches.
   • By analyzing the pattern of proteolytic cleavage using techniques such as SDS-PAGE or mass spectrometry, hydrophobic patches can be mapped.

These experimental methods provide valuable information about the hydrophobic patches on a protein and can aid in understanding the protein's structure, function, and interactions with other molecules. Multiple approaches may be used in combination to gain a comprehensive understanding of the hydrophobic regions of the protein.