Turning a PAGE on Protein Dynamics

By Jason Socrates Bardi

Studying protein structure and dynamics is not what it used to be.

In the last several decades, primary, secondary, tertiary, and quaternary structure determination have all become routine due to advances in the techniques and technologies of molecular biology and spectroscopy. Molecular Biology Professor Jack Johnson at The Scripps Research Institute, for instance, has been using x-ray crystallography to solve the structures of intact complex virus particles for over a decade.

So what about observing the motions of intact complex virus particles biochemically?

"Usually, you don't think about being able to characterize the motions [of a molecular machine] biochemically," says Johnson. "You need spectroscopy or something that is going to be time-sensitive."

However, in the latest issue of Molecular Cell, Scripps Research graduate student Lu Gan, Johnson, and their colleagues report the individual steps of the maturation of a virus called bacteriophage HK97, which they elucidated with a highly unusual application of a routine laboratory technique called SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

A Virus that Plagues Bacteria

The bacteriophage HK97 is a double stranded DNA virus that infects Escherichia coli cells. It was first isolated from pig dung by a rice geneticist in Hong Kong.

The mature virus particles have a hard protein head, the "capsid," and a long protein tail. In nature, these viruses infect the bacteria by attaching their tails to bacterial cell walls and injecting in their DNA. Once inside, the DNA will circularize, use the bacterial enzymes to make copies of its DNA, replicate its proteins, assemble new virus particles, and eventually lyse the cell, spilling out new viral particles.

When the viral subunits assemble, they first come together to make an immature, round procapsid called "prohead." Then, when all the proteins are in place and the DNA is ready to be packaged into the prohead, a viral protein complex sitting at a vertex (called the portal) of the virus pumps in the DNA. While this is happening, the prohead matures into its final form.

Maturation is an important part of the viral lifecycle. When the viral capsid matures, which takes about five minutes in nature, it goes from being a round particle to an icosahedral particle with angular vertices and distinct triangular faces. It also expands from a diameter of about 560 Angstroms to a diameter of about 660 Angstroms (an Angstrom, one billionth of a centimeter, is a common yardstick for molecular-scale objects) While 100 Ang isn't considered a large change at the macroscopic scale, at the length scale of the entire virus, the average diameter has increased nearly 20 percent, and the internal volume has nearly doubled.

At the same time, the capsid forms covalent cross-links between particular lysine and asparagines amino acids within 420 different protein subunits that make up this complex protein shell. These cross-links serve to permanently bind the subunits of the capsid to each other.

The mature and immature forms of the virus are identical in composition, but completely different in size and stability. In the final form, almost every subunit has changed its position, and all the cross-linking serves to tighten the structure of the viral head.

"The proteins are totally reorganizing," says Johnson. "When they link together, they form what we call chain mail."

The chain mail is extremely durable and makes the virus more chemically and mechanically stable. It helps protect the DNA during transport from one host to another. Once the DNA is inside and all the linkages are in place, the tail is attached and the virus is mature and ready to infect another E. coli cell. As a testament to this stability, the only known method to proteolyze the mature capsid is to heat it up to greater than 65 degrees Celsius and then hit it with a thermophilic protease.

"We think of this [viral capsid] as a molecular machine," says Gan. "Its goal is to expand into the mature state."

Using SDS PAGE, Gan observed the individual steps of this maturation process.

A New Page for PAGE

SDS PAGE is not something that one normally regards as a powerful technique for analyzing details of protein structure. It is more a routine assay used to determine crude measurements of rough molecular weights or purity of a protein solution. Watching the dynamics of a system of interacting molecules is normally a job reserved for sensitive techniques like nuclear magnetic resonance or time-resolved electron microscopy. Divining fine details about protein quaternary structure with SDS PAGE should be like smashing a music box with a hammer and spreading all the pieces out on a table to figure out what songs it plays—something too crude to work.

The SDS PAGE technique begins with mixing the protein solution with a strong detergent that completely denatures (unfolds) the proteins. This solution is then run through a sieve-like gel, which separates all the proteins in the mixture by virtue of size or weight. Then the gel is stained and dried, and finally it shows "bands" of distinct proteins or protein fragments separated by weight.

But since the subunits of the HK97 virus link covalently as the viral head expands into its final form, SDS PAGE gave Gan a perfect way to resolve the individual pieces of the capsid as the reaction proceeded and to read out how many of the linkages were in place at various points in the reaction.

Each time a cross link was made within the viral capsid, a new band would appear on the gel, and this allowed them to delineate all the individual steps of viral maturation. Thus, they were able to keep track of the motions of this molecular machine using simple biochemistry.

"As far as we know," says Johnson, "there are not really any [other studies] like this."

Part of a Larger Collaboration

The paper is a collaboration between Johnson's group and several other scientists around the world. Johnson's long-time collaborator Robert L. Duda, of the University of Pittsburgh and the Pittsburgh Bacteriophage Institute, is the corresponding author on the paper and one of the world's leading experts on these types of viruses. A few years ago, Duda discovered a way to express the proteins that make the head of the virus in such a way that they can assemble and mature without the DNA being present.

Duda and his colleague Brian Firek, who is also an author on the paper, refined an experimental setup that was used in the current study and carefully quantified results from gels with the data from an older paper. Doing so, they were able to elucidate the "point of no return" for the maturing virus—the maturation state that can only proceed in a forward direction and cannot revert to prohead. Usually, it is impossible to study maturing states of a virus biochemically because the intermediate states have such a fleeting existence. But HK97 intermediates biochemically trapped at low pH so that they have lifetimes on the order of hours to days, which allows them to be scrutinized with "slow" techniques like SDS-PAGE.

Gan developed the conditions to drive it out of the prohead state into a nearly mature state, and he characterized the chemistry that takes place as this maturation occurs. Duda provided the guidance for the study and is joined on the paper by his colleague Roger Hendrix, also of the University of Pittsburgh and the Pittsburgh Bacteriophage Institute.

Johnson and Duda have been collaborating since 1995, and they originally solved the high-resolution structure of the mature head state of the HK97 viral capsid. In a later paper reported a low-resolution cryo-EM structure of the prohead, into which was fitted the atomic coordinates of the mature Head. The fit was so good that it became clear the virus matures by some form of rigid body rotations and local refolding. The results of these two studies gave the scientists the idea that parts of the capsid subunits must move a relatively long distance—up to 35 Angstroms—in order to achieve one cross-link.

"Lu came to this laboratory to do a rotation," says Johnson. This paper is the result of over a year's worth of work that was a tangent to this original project, and it has grown to become somewhat larger than the original project from which it sprang.

At the time, Johnson and his colleagues thought that the process of cross-linking within the virus capsid was much simpler and something that happened after the capsid had expanded into its final form.

"Lu showed that there was this entire complex process [whereby the virus expanded and cross-linked]," says Johnson. "And he teased out all the molecular details and has been able to use chemistry to monitor the transitions."

Gan showed that the amino acids in the subunits of the virus head cross-linked to each other throughout the transition from immature to mature virus (instead of at the end, as originally assumed), and was able to show which subunit linked to which and the order in which the linking occurred.

Also collaborating on the paper was Alasdair C. Steven of the Laboratory of Structural Biology National Institute of Arthitis and Muskuloskeletal and Skin Diseases at the National Institutes of Health and James F. Conway of the Institut de Biologie Structurale in Genoble, France.

Conway and Steven applied a powerful structural biology technique called cryo-electron microscopy to a maturation intermediate state of the maturing viral capsid and verified that what Gan had discovered was correct.

In addition to shedding light on basic questions such as how protein assemblies change their shape, interact with other proteins, and assemble themselves, the work is important because the HK97 virus has properties similar to some animal viruses, particularly herpes viruses. In fact, the vast majority of complex viruses change their morphology and shape as they mature. Like HK97, the herpes procapsid morphology is round, while the capsid is angular. Herpes also packages its DNA similarly.

This work may lead to better understanding of the maturation mechanisms of herpesvirus and other human viruses and may lead to ways to address the diseases they cause.

Next: High-Resolution Structures

At the moment, Gan and Johnson are working on making crystals of the nearly mature form of the HK97 virus head so that they can solve the high-resolution structure, compare it to the immature prohead and mature head structures, and really investigate the mechanism of the virus capsid's maturation on the atomic level.

Johnson quips that, even though Gan is halfway towards his thesis by now, he would still like to see Gan complete the original aim of his rotation to crystallize this expansion intermediate form of the capsid—a process that involves subjecting purified virus particles to a myriad of different salt and buffer concentrations until the exact combination can be found that allows the virus to array symmetrically in solution to form a crystal. Only then will a beam of x-rays diffract off the crystal in a pattern that can be collected, refined, analyzed, and resolved into a three-dimensional picture of the viral structure.

"We'd like to study the mechanism by crystallography," says Johnson, joking that his laboratory focuses on crystallography and he would like Lu to come out of his lab with some crystallography experience.

All kidding aside, Johnson says, Lu has done a wonderful job. "This is what quality investigation is all about," he says.

To read the article, "Control of Crosslinking by Quaternary Structure Changes during Bacteriophage HK97 Maturation" by Lu Gan, James F. Conway, Brian A. Firek, Naiqian Cheng, Roger W. Hendrix, Alasdair C. Steven, John E. Johnson, and Robert L. Duda, see the June 4, 2004 issue of the journal Molecular Cell or go to http://www.molecule.org.

Send comments to: jasonb@scripps.edu

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Professor Jack Johnson (right) says graduate student Lu Gan has done a wonderful job. Photo by Kevin Fung.