Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins.

Live-cell super-resolution imaging of actin using LifeAct-14 with a PAINT-based approach.

We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells.

PAINT using proteins: A new brush for super-resolution artists.

PAINT (points accumulation for imaging in nanoscale topography) refers to methods that achieve the sparse temporal labeling required for super-resolution imaging by using transient interactions between a biomolecule of interest and a fluorophore. There have been a variety of different implementations of this method since it was first described in 2006. Recent papers illustrate how transient peptide-protein interactions, rather than small molecule binding or DNA oligonucleotide duplex formation, can be employed to perform PAINT-based single molecule localization microscopy (SMLM).

LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions.

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein.

A threonine zipper that mediates protein-protein interactions: Structure and prediction.

We present the structure of an engineered protein-protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close-packing of the Thr residues, with only one intermonomer hydrogen bond (H-bond) between two of the Thr residues. This Thr-rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues.

Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces.