World first protein crystallisation images
Image: Shell growth in Caulobacter crescentus. [Colin Comerci, Jonathan Herrmann/Stanford University]
In a world first, researchers have imaged proteins assembling into crystals, one molecule at a time, in a living cell.
Using super-resolution fluorescence microscopy and single-molecule tracking, Nobel Laureate, Professor William E Moerner from Stanford, and colleagues, watched as individual protein molecules moved around the surfaces of living bacteria to form a shell.
“I’ve been super-excited to watch and track the movements of single molecules as they form this fascinating crystalline shell on the surface of a microbe,” says Moerner. “We can look on a very fine scale and see the molecules arranging themselves in the shell. It’s the first time we’ve been able to do this.”
First observation ever made of protein crystallization by a living cell. Single protein molecules (red) traverse the surface of a microbe over the course of two minutes, joining an existing patch of the microbe’s shell (green) to crystallise. The molecules are tagged with fluorophores. [Josh Yoon, Colin Comerci, Jonathan Herrmann/Stanford University]
The bacterium, Caulobacter crescentus, lives in lakes and streams, and secretes RsaA protein building blocks to form a thin crystalline shell, known as a surface layer or S-layer.
To study protein crystallisation as it took place, the researchers first stripped microbes of the S-layers and then supplied the bacteria with modified, fluorescently-labelled RsaA building blocks.
They then tracked the glowing proteins as a shell was formed, covering the microbe in a hexagonal, tile-like pattern in less than two hours.
Using live cell stimulated emission depletion (STED) microscopy they could observe structural details of the layer down to 60 nm.
The researchers discovered that crystallisation didn’t take place as they had anticipated.
Rather than being guided into position and joined to the shell by enzymes as takes place in most biological reactions, the shell forms via 'topologically guided continuous protein crystallisation'.
Illustration shows how protein building blocks secreted by a microbe (at arrows) travel over its surface until they encounter its growing crystalline shell. There they join one of the six-sided units that tile the microbe’s surface. [Greg Stewart/SLAC National Accelerator Laboratory]
Here, a protein building-block traverses the surface of the microbe until it reaches a patch of crystalline shell, at which point it joins one of the pre-existing six-sided units that cover the microbe's surface.
As the researchers point out in Nature Communications, the surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly.
Still image shows the tracks (red, white and blue lines) of individual protein molecules moving around the surface of a microbe over a period of 60 seconds. One of the molecules has just bound to an existing patch of the shell (bottom), which is labelled with a green fluorescent tag. The microbe is outlined in orange. [Josh Yoon, Colin Comerci, Jonathan Herrmann/Stanford University]
“The protein molecules are self-assembling building blocks, and they will spontaneously form themselves into crystals,” says Jonathan Herrmann, a PhD student at Stanford and the Department of Energy’s SLAC National Accelerator Laboratory. “No enzyme is required.”
A closer look at areas where shell growth is occurring: green areas are existing patches of shell; red areas are new growth at cracks, the ends (poles) of the microbial cell and in the middle, where the microbe is growing and preparing to divide. [Colin Comerci, Jonathan Herrmann/Stanford University]
As Professor Soichi Wakatsuki from Stanford and SLAC points out, this new way of observing shell formation is opening up a new way to understand and eventually manipulate surface layer structures, both in living organisms and in isolation.
“Now that we know how [the shells] assemble, we can modify their properties so they can do specific types of work, like forming new types of hybrid materials or attacking biomedical problems,” he says.
The researchers will now use higher resolution X-ray and electron imaging at SLAC to investigate the shell crystallisation process further.
Research is published in Nature Communications.