Our understanding of the microscopic world has been shaped by the tools available to monitor and visualize cellular interactions. We “stand on the shoulders of giants” to propel our research to even greater heights. Studying protein-protein interactions (PPI) has proved fruitful for our understanding of cellular metabolism, signal transduction, and more. Scientists are starting to build whole organism interactomes (kindred to the metabolome and genome) that could have huge implications towards understanding and treating disease. Let us take a trip down memory lane to see where we have come from.
1894 – Fundamental Isolation
Albrecht Kossel is considered a pioneer in genetics research. The methods employed may seem simple by today’s standards, but through purification and precipitation he was able to isolate nuclei from the red blood cells of geese. Further chemical analysis of the nuclear extract led Kossel to believe he discovered a new class of proteins, which he termed histones. They appeared to be bound to nucleic acids because they were soluble in neutral water and precipitated upon adding sodium chloride. Kossel further characterized nucleic acids and discovered the five basic nucleotides: adenine, cytosine, guanine, thymine, and uracil.
1925 – Ultracentrifuge
Studying physical properties of colloids included sedimentation analyses, for which Theodor Svedberg constructed the ultracentrifuge. This advanced Kossel’s precipitation studies by providing a higher level of separation between molecules of different sizes and solubilities.
1962 – Green Fluorescent Protein
The discovery, isolation, classification and modification of green fluorescent protein (GFP) by scientists Osamu Shimomura, Martin Chalfie, and Roger Tsien earned them a joint Nobel Prize in 2008. This tool has enabled countless scientists to investigate previously invisible phenomena inside cells. The GFP gene can be linked to a particular gene of interest and used as a reporter. If the target gene is expressed, then so too will GFP be expressed. Shining UV or blue light on the cells will result in the GFP molecule fluorescing green and thus indicating that the target gene is expressed. Numerous color mutants of GFP have since been created allowing for a more comprehensive picture of protein expression.
1989 – Two-Hybrid Screening
The yeast two-hybrid system described by Stan Fields and Ok-Kyu Song was the gold standard for PPI studies for many years. It takes advantage of transcription factors that have separate binding and activating domains. The two domains are first bound to target proteins, and then if there is interaction between them, the reporter gene is expressed and detected.
1989 – Electrospray Ionization
John Fenn and colleagues demonstrated the use of mass spectrometry to study large, polar molecules. Electrospray ionization results in distinct spectrums of peaks which are able to classify large protein interactions. Similar biophysical techniques include surface plasmon resonance (SPR) instruments, first manufactured by Pharmacia in 1984, and nuclear magnetic resonance (NMR) instruments, first manufactured by Varian in 1949.
New Methods for PPI Research
The number of breakthroughs that advanced protein research could certainly extend well beyond this list. Scientists have been able to characterize stable protein interactions using many of these methods, yet as we advance towards whole-organism interactomes, we realize there are many gaps in our understanding. Transient protein interactions are a major area of focus for scientists because they play a crucial role in regulation and function inside the cell.
Some of the most promising new technologies to measure transient PPI use fluorescence and bioluminescence. Sensitivity is usually a major deciding point in assay selection, and luminescence provides the advantage of greater signal-to-background ratio than fluorescence. Other considerations include steric hindrance, self-affinity, and reagent stability. Andrew Dixon et al. recently described a new luciferase-based system, NanoBiT, which offers new capabilities for real-time analysis of protein interactions in live cells, including the ability to detect interactions at low levels of expression.
Learn more about NanoBiT technology here.