His conclusion? Proteins are very tolerant. “Many people had the idea that a protein structure is very complicated—that there’s very fine balance between the unfolded and the folded protein,” says Matthews. “People believed that if you just randomly made substitutions here and there you would probably prevent folding or at least seriously compromise the protein.” Matthews found the contrary: He made multiple substitutions at many sites and the three-dimensional structure was not changed at all. The amino acids on the surface of the protein were especially forgiving—Matthews changed many of them and the protein remained active.

David Agard, an HHMI investigator at UCSF, tackled the protein-folding problem with a different technique—NMR spectroscopy. This powerful method is useful for looking at smaller proteins in buffer solutions. The molecules are exposed to a giant magnet that causes their nuclei to absorb and re-emit electromagnetic radiation. This emitted energy gives clues about each atom’s orientation and location in the protein.

Agard chose a small bacterial enzyme, called alpha-lytic protease, to look at the different shapes a protein assumes as it folds into its final active form. Normally, these intermediate structures are difficult to examine because they’re so unstable and transient. But Agard discovered that if he cut off a piece of the enzyme called the proregion, the protease would be frozen in an intermediate form, which could easily be visualized with NMR spectroscopy. His experiments revealed a surprise: The proregion helps alpha-lytic protease fold into a very unstable, high-energy form before assuming its final active shape.

Not ones to shy away from new technology or big projects, Agard and his UCSF colleague and then HHMI investigator John Sedat also developed several microscopy techniques to help them look at protein machinery at work inside cells.

“Our HHMI support allowed us to make leaps in technology that wouldn’t have been possible any other way,” says Agard. “At the time, the electron microscopists generally weren’t doing big complex cellular things. So we had to combine methodologies and formulate new strategies for collecting and processing data in three dimensions.”

One of the techniques they pioneered was cryoelectron tomography, which involves flash-freezing a cell, photographing it from different angles, and combining the photos to create a three-dimensional model of the cell’s contents. Agard and Sedat used this technique to examine a molecular complex called the centrosome, which is responsible for ensuring that equal numbers of chromosomes are distributed to the mother and daughter cells during cell division. Their images revealed just how the centrosome goes about organizing microtubule fibers in the cell and how the chromosomes then line up on the fibers and move to their respective ends of the dividing cells.

The Centrosome
David Agard and John Sedat used cryoelectron tomography to examine the centrosome and come up with models like this one for how microtubules organize during cell division.
Credit: Janet Iwasa / One Micron.com

The Shape of Things to Come

Today, there are 36 investigators at 24 institutions in the HHMI structural biology program. More than 86,000 protein structures have been solved and submitted to the Protein Data Bank, an international repository for structural data. “Right now, we are at the point where the techniques have become almost perfect,” says Deisenhofer. “As soon as you have a crystal you can almost certainly determine the structure in a relatively short amount of time. It’s become a standard technique in many laboratories.”

In the coming years, emerging technologies are likely to increase the number and type of molecules that can be studied. For example, a team of scientists led by HHMI investigator Axel Brunger at Stanford University is using a free-electron laser that shoots x-rays at very small crystals of proteins that are hard to crystallize (membrane proteins, for example), opening up a world of structures that had been off-limits for x-ray analysis.

Then there’s the new trend of combining techniques to look at larger assemblies, like Agard and Sedat did. For example, Steitz, who shared the 2009 Nobel Prize in Chemistry for solving the structure of the ribosome—a huge complex of RNA and protein—is blending cryoelectron microscopy and crystallography to capture snapshots of the ribosome as it goes about its job transcribing proteins.

“Between cryoelectron microscopy and the free-electron laser, I imagine we won’t need crystals at all,” says Agard. “The combination of these tools will make it so that we understand the structure of all the molecular complexes at high resolution in isolation and at moderate resolution in the context of the cell. The ability to look at how these complexes are interacting, how they interchange, and what their dynamics are, that’s where I imagine things going.”

Part 1 of 2. In the next issue, our series on HHMI’s structural biology program continues with a look at some of the research that is coming out of the program.

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