When you think of proteins — the enzymes, signaling molecules, and structural components in any living thing — you might think of single strands of amino acids, organized like beads on a string. But almost all proteins consist of multiple strands folded and linked together, forming complicated 3D superstructures called molecular assemblies. One of the key steps to understanding biology is figuring out how a protein does its job, which requires knowledge of its structures down to the atomic level.
Over the past century, scientists have developed and used amazing technologies such as X-ray crystallography and cryo-electron microscopy to determine the structure of proteins, thereby answering countless important questions. But the new work shows that understanding the structure of proteins can sometimes be more complicated than we think.
A group of researchers from Lawrence Berkeley National Laboratory (Berkeley Lab) studying the world’s most abundant protein, an enzyme involved in photosynthesis called rubisco, showed how evolution can lead to a surprising variety of molecular assemblies that all perform the same task. The findings, published today in Advances in sciencereveal the possibility that many of the proteins we thought we knew actually exist in other, unknown forms.
Historically, if scientists picked a structure and determined that a protein was dimeric (made up of two units), for example, they could assume that similar proteins also existed in a dimeric form. But small sample size and sampling bias — inevitable factors given that it is very difficult to convert naturally occurring liquid proteins into solid, crystallized forms that can be examined via X-ray crystallography — were the obscuring reality.
“It’s like going out and seeing someone walking their dog, if you’d never seen a dog before, then you’d see a wild dog, you’d think, ‘OK, that’s what they all look like. dogs”. But what you have to do is go to the dog park and see all the diversity of dogs that is there,” said lead author Patrick Shih, a faculty scientist in the Biosciences Area and Director of Plant Biosystems Design at The Joint Institute. of BioEnergy (JBEI). “One point from this paper that goes beyond rubisco to all proteins is the question of whether or not we’re seeing the true range of structures in nature, or are these biases that make everything look like a dog wild.”
Hoping to explore all the different arrangements of rubiscos in the metaphorical dog park and learn where they came from, Shih’s lab collaborated with the Bioscience Area’s structural biology experts using Berkeley Lab’s Advanced Light Source. Together, the team studied a type of rubisco (form II) found in bacteria and a subset of photosynthetic microbes using traditional crystallography — a technique capable of atomic-level resolution — combined with another structure-solving technique, with small-angle X-ray scattering (SAXS), which has lower resolution but can take pictures of proteins in their native form when they are in liquid mixtures. SAXS has the added advantage of high-throughput capability, meaning it can process dozens of separate protein assemblies in sequence.
Previous work had shown that the best-studied type of rubisco found in plants (form I) always takes an “octameric core” array of eight large protein units arranged with eight small units, while form II was believed to exist mainly as a dimer with some rare examples of six-unit hexamers. After using these complementary techniques to examine rubisco samples from a diverse range of microbial species, the authors observed that most form II rubisco proteins are actually hexamers, with random dimers, and they discovered a previously unseen tetrameric (four units) assembly.
Combining this structural data with the corresponding protein-coding gene sequences allowed the team to perform ancestral sequence reconstruction — a computer-based molecular evolution method that can estimate what ancestral proteins looked like based on the sequence and appearance of modern proteins that evolved from them. .
The reconstruction suggests that the gene for form II rubisco has changed during its evolutionary history to produce proteins with a variety of structures that transform into new forms or revert to old structures quite easily. In contrast, over the course of evolution, selective pressures led to a series of changes that locked the I rubisco form into place — a process called structural imprinting — which is why octameric assembly is the only arrangement we see now. According to the authors, it was assumed that most protein assemblies became entrenched over time by selective pressure to improve their function, as we see with form I rubisco. But this study suggests that evolution may also favor flexible proteins.
“The big finding from this paper is that there is a lot of structural plasticity,” said Shih, who is also an assistant professor at UC Berkeley. “Proteins may be much more flexible, across the board, than we’ve been led to believe.”
After completing the reconstruction of the ancestral sequence, the team performed mutational experiments to see how changing the assembly of rubisco, in this case breaking a hexamer into a dimer, affected the activity of the enzyme. Unexpectedly, this induced mutation produced a form of rubisco that is better at using its target molecule, CO2. All natural rubiscotes often bind O of similar size2 molecule in the accident, reducing the productivity of the enzyme. There is great interest in the genetic modification of rubisco in agricultural plant species to increase the affinity of the protein for CO2, in order to produce more productive and resource-efficient crops. However, there has been much focus on the active site of the protein—the region of the protein where CO2 or O2 connect
“This is an interesting insight for us because it suggests that to have more fruitful results in rubisco engineering, we can’t just look at the simplest answer, the region of the enzyme that actually interacts with CO.2” said first author Albert Liu, a graduate student in Shih’s lab. “Maybe there are mutations outside of that active site that actually participate in this activity and could potentially change the function of the protein in the way we want. So this is something that really opens the door to future avenues of research.”
Co-author Paul Adams, Associate Director of the Biosciences Laboratory and Vice President for Technology at JBEI added, “The mix of techniques used and the interdisciplinary nature of the team was a real key to success. The work highlights the power of combining genomic data and methods of structural biology to study one of the most important problems in biology and reach some unexpected conclusions.”
The structural biology experiments were performed at Berkeley Lab’s Advanced Light Source (ALS), a Department of Energy (DOE) Office of Science user facility. The SYBILS beam is funded in part by DOE’s Office of Biological and Environmental Research. X-ray crystallography was performed at the Berkeley Center for Structural Biology. JBEI is a Bioenergy Research Center managed by Berkeley Lab. This work was funded by the DOE Office of Science and the David and Lucile Packard Foundation.