Highly Selective Fluorescent Biosensor for Labeling His-Tagged Proteins

The Solution

The biosensor presented here is comprised from three main molecular-components: (I) the selective binder (binds to a specific tag, e.g. His-tag); (II) the nonselective binder (binds to the protein surface, as long it is in a specific conformation); and (III) a solvatochromic fluorophore.

The selective binder (I) ensures the sensor will only bind to a tagged protein (in this case, His-tagged protein), regardless of its conformational state. Once the protein folds into the specific conformation, the nonselective binder (II) detects and binds to the modified surface configuration (pre-designed to detect the specific conformation). Now that the local environment of the attached-biosensor and its fluorophore (III) has changed, the fluorophore subsequently emits a distinct and measurable signal.

An example of a protein-surface sensor consisted of a tri-Ni2+-NTA complex (I), a tripodal peptide receptor (II), and a dansyl group (III), which serve as a His-tag binder, a protein-surface receptor, and a solvatochromic probe, respectively.

Advantages

• Directly, rapidly, and easily applicable.
• Usable in complex biological environments live cells or biological media.
• Demonstrates high affinity and superior signal-to-noise ratio.
• Small in size - unlikely to interfere with the labeled protein function.
  

Applications

• Fluorescent detection of specific conformations of His-tag labeled proteins.
• Direct labeling in both live cell imaging and biochemical assays.
• Tracking protein-protein interactions in vivo.
• Localizing proteins and measuring their steady-state concentration in vivo.
• High throughput screening of inhibitors of protein-protein interactions in living cells

Development Status

The methodology of creating fluorescent molecular sensor responding to changes occurring on the surfaces of specific proteins was successfully demonstrated (Published in: Chem. Eur. J. 2015, 21,15981 [1]). The basis of this technology was later demonstrated in living cells expressing his-tagged proteins showing effective and reversible operation. Other project expanding the applications of this invention are currently in progress. Two filed patent applications cover this invention.