The Protein Printer
Page 2: Frozen in a flash
As the prefix suggests, samples for cryo-electron microscopy must first be cooled – or rather flash frozen. Ribosomes or ribosomal complexes, painstakingly isolated from nucleated or bacterial cells, are placed on a thin carbon film deposited on a copper grid and frozen by plunging them into liquid ethane. The nanomachines are instantaneously captured while they are at work and can be subsequently imaged by electron microscopy. Moreover, the micrographs can be interpreted as snapshots of the process of protein synthesis. Because the glass-like solid in which they are immobilized is vitrified ice which (unlike normal ice) contains no crystals, the fragile and complex architecture of the ribosomes is preserved in such detail that the researchers can reconstruct the sequence of steps involved in protein production and view it like a time-lapse movie. “Depending on the protein and organism involved, synthesis can take anywhere from a few seconds to several minutes,” says Beckmann. “We want to observe the crucial transition stages.”
Novel direct electron detectors have revolutionized the practice of cryo-electron microscopy. Researchers call it the ‘resolution revolution’, because it has greatly enhanced the resolution of the micrographs, to a few tenths of a nanometer. Visitors to Beckmann’s department in the Gene Center in Grosshadern can observe not only the preparation of samples in the lab, but join his research staff as they inspect the data being acquired by the electron microscope. Some screens are filled with columns of numbers; some researchers are analyzing the micrographs themselves. It is even possible to switch to what the detectors are “seeing”. These “live” pictures are normally shown on the big monitor in the department’s kitchen. These provide the unprocessed data that form the basis for the high-resolution two-dimensional images, taken from different angles, which are used to construct three-dimensional views. In fact, the investigators can actually zoom in to study the finer details.
Integration into the cell membrane
Beckmann is particularly interested in one particular component of the cells of higher organisms – the Sec61 complex or translocon, onto which ribosomes can dock. The translocon acts as a conduit for proteins and plays a key role whenever proteins need to be inserted into or transported through a membrane. About one-third of all proteins are either inserted into the cell membrane where they serve as signal receptors, Beckmann explains, or are secreted by the cell, to act as of antibodies or digestive enzymes, for instance. Beckmann’s group has used structural analysis to decipher how the so-called ribosome-translocon complex orchestrates the passage of proteins into and through membranes. The Sec61 complex itself forms a molecular channel into which the ribosome directs the growing protein. Beckmann determined the precise structure of this channel last year. The findings revealed that the ribosome binds directly to the translocon on the membrane and feeds the nascent protein into the channel, and either into the membrane or straight through it. In order to understand how the process works, it is necessary to analyze the structure of the whole complex in as many functional states as possible.
For proteins destined to be integrated into membranes or secreted to the outside, the ribosome needs to know the correct destination of each. Günter Blobel discovered that proteins carry a kind of postal code at the front end of the amino-acid sequence, a zip code that can be read by the proteins of the cell’s routing system. This short sequence ensures that proteins are piloted to the right location. The idea was initially met with skepticism, but Blobel, one of the “original gangsters” (Beckmann’s term) in the field, turned out to be right. The basic principle was found to be generally valid, and functions in the same way in yeasts as in plant and animal cells. And Blobel won the 1999 Nobel Prize for demonstrating it. Beckmann was a postdoc in Blobel’s lab at the time and followed the awards ceremony live. “It was as if a tsunami had hit the lab,” he recalls.
Ribosome research has experienced a boom in recent years, but many of the organelle’s functions remain mysterious. For instance, Beckmann would love to know how the protein factories carry out quality control. Clearly, they are equipped to detect errors in the blueprint (i.e., the mRNA), and can assess whether the growing protein is functional. “There is nothing more damaging for an organism than the accumulation of defective proteins encoded by corrupted mRNAs,” Beckmann says, because this can lead to the loss of essential functions. But whenever a ribosome reaches the end of an mRNA without having encountered a termination codon, for instance, it “knows” that the protein is incomplete – because it cannot be released. The stalled ribosome then recruits specialized release factors that detach it from the defective mRNA and its protein product, and ensure that both are degraded.
A class of cellular apps
Beckmann has been studying ribosomes for nearly two decades, and he speaks of these – in molecular terms – huge complexes, with dimensions of up to 35 nm, with something like respect. Ribosomes are not perfect, but they are extremely versatile machines, he says. Only very recently, researchers realized that, in addition to their primary task, they also have many part-time duties, in relation to quality control, for example. It turns out that an array of accessory proteins can bind to the surface of the ribosome to facilitate the recognition and translation of codons in the mRNA, and monitor and promote the growth of its protein products. Each factor is like an app that enables the ribosome to do something amazing, Beckmann says. “For example, they can measure the forces acting on cell components, and estimate amino acid and antibiotic concentrations. The spectrum of specialties is broad and varies from organism to organism.”
Beckmann is not only interested in how the molecular machines work. His team is now looking at what happens to the ribosome when it comes off the job. Essentially, the organelle dissociates into its two subunits, which are then ready for the next round of synthesis. “Only recently have we learned that, in eukaryotic cells, the enzyme ABCE1 has a major influence on this step,” Beckmann explains. Basically it uses a lever to force the subunits apart, and since ABCE1 contains clusters of bound iron, Beckmann refers to this as ‘the iron fist’. Members of his group are now trying to find out how this essential step in protein synthesis works in structural terms. “This sort of work can take months or years,” he adds.
It is conceivable that the future may provide us with a completely new picture of the mechanisms involved in protein synthesis. “Very often, in pursuit of one goal, we stumble across something unexpected that we also need to understand,” says Beckmann. It looks as if he’s unlikely to run out of puzzles for him and his coworkers to solve in the coming years. “At all events,” he avers, “ribosomes have a few more surprises in store for us.”
Prof. Dr. Roland Beckmann
Professor of Biochemistry at the LMU Gene Center. Beckmann, born in 1965, studied Biochemistry at the Free University in Berlin, where he did his PhD before moving to Rockefeller University in New York in 1995 as a postdoc. He later led his own research group at the Charité Hospital at Berlin’s Humboldt University, before being appointed as professor at LMU in 2006.