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. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

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. We envisage a similar approach could be applied to imaging other proteins within live mammalian 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). We discuss the different approaches to PAINT using peptide and protein interactions, and their applications in vitro and in vivo. We highlight the important parameters to consider when selecting suitable peptide-protein interaction pairs for such studies. We also note the opportunities for protein scientists to apply their expertise in guiding the choice of peptide and protein pairs that are used. Finally, we discuss the potential for expanding super-resolution imaging methods based on transient peptide-protein interactions, including the development of simultaneous multicolor imaging of multiple proteins and the study of very high and very low abundance proteins in live cells.

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. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion 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. In previous work, we have shown that the side chain (χ1 ) dihedral angles of interface and core Thr residues can be predicted with high accuracy using a hard sphere plus stereochemical constraint (HS) model. Here, we demonstrate that in the Thr-rich local environment of the Thr zipper structure, we are able to predict the χ1 dihedral angles of most of the Thr residues. Some, however, are not well predicted by the HS model. We therefore employed explicitly solvated molecular dynamics (MD) simulations to further investigate the side chain conformations of these residues. The MD simulations illustrate the role that transient H-bonding to water, in combination with steric constraints, plays in determining the behavior of these Thr side chains. Broader Audience Statement: Protein-protein interactions are critical to life and the search for ways to disrupt adverse protein-protein interactions involved in disease is an ongoing area of drug discovery. We must better understand protein-protein interfaces, both to be able to disrupt existing ones and to engineer new ones for a variety of biotechnological applications. We have discovered and characterized an artificial Thr-rich protein-protein interface. This novel interface demonstrates a heretofore unknown property of Thr-rich surfaces: mediating protein-protein interactions.

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. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic.