by Rebecca Kolberg
RNase H domain of HIV-1 reverse transcriptase
As members of the Protein Expression Laboratory (PEL) can attest, when it comes to intramural research at NIH, movement may not necessarily mean packing up your bags and physically moving to another spot. By this fall, PEL should have a new administrative home, some much-needed new staff, and some new avenues of scientific exploration.
Since it opened in August 1990, PEL has operated under the Office of the Director, charged with producing recombinant human immunodeficiency virus (HIV) proteins and related proteins needed by intramural scientists. However, because OD lacks authority and personnel mechanisms to train biomedical researchers, PEL has not been able to bring aboard scientific staffers crucial to the function of most NIH labs: postdocs.
To solve this problem, and to get PEL into a more simpatico intellectual and administrative home, plans are now in motion to move PEL from OD to NIAMS. After reviewing a number of proposals from several intramural programs, Deputy Director for Intra-mural Research Michael Gottesman decided that the NIAMS proposal was most likely to ensure PEL's continued service to the NIH community. Currently, the OD and NIAMS are negotiating a memorandum of understanding guaranteeing that PEL will remain a resource for all intramural researchers. Other factors entering into the choice of NIAMS as PEL's new home were the lab's proximity to NIAMS labs in Building 6, the desire to strengthen NIAMS' intramural research presence, and the synergy of PEL with research programs within NIAMS.
Many NIH scientists who concentrate on HIV-related research value the resources and services provided by PEL, which is headed by Paul Wingfield and Stephen Stahl. The protein "factory," which keeps small amounts of a half-dozen or so HIV proteins in its freezer, is capable of churning out up to 1 g of protein with its Escherichia coli expression system. However, if the HIV protein in question is readily available commercially, such as HIV-1 protease, PEL prefers to steer researchers to the appropriate supplier through the NIH AIDS Research and Reference Reagent Program Catalog run by the Office of AIDS Research.
Supplying the proteins and expertise needed to meet intramural research demands has kept PEL's six-person staff more than busy over the past five years. Prominent intramural groups, including those headed by Angela Gronenborn, Marius Clore, and Ad Bax of NIDDK and Alasdair Steven of NIAMS, have relied heavily on recombinant proteins produced, purified, and characterized by PEL for X-ray crystallography and high-resolution nuclear magnetic resonance (NMR) spectroscopy studies of potential targets for HIV therapies or vaccines.
But other intramural researchers -- whose work only occasionally or tangentially relates to HIV -- may not be aware that PEL researchers not only make HIV proteins, but also make "house calls" providing scientists with consultation on the expression and purification of recombinant HIV and related proteins within their own labs.
Projects to which PEL collaborators have made substantial contributions have addressed a wide range of scientific questions. "They [PEL scientists] have almost a preternatural ability to advance a large number of projects simultaneously," says Steven, chief of the Laboratory of Structural Biology at NIAMS.
Since 1990, Wingfield, Stahl, and their colleagues have played an instrumental role in the purification, characterization, and analysis -- using innovative 15N relaxation NMR spectroscopy measurements -- of the ribonuclease H domain of HIV-1 reverse transcriptase. Wingfield, the laboratory's chief and its expert on protein structure, co-authored a study with Clore and Gronenborn published in the May 21, 1993, issue of Science on the kinetics of the folding of the large, all-beta sheet protein, interleukin-1[[beta]] -- considered to be one of the most difficult protein-folding problems solved to date. Stahl, head of the lab's molecular biology section, soon followed suit, co-authoring a paper with Clore and Gronenborn in the July 23, 1993, issue of Science that details the NMR structure of a complex between the DNA-binding domain of the chicken erythroid transcription factor GATA-1 and its target DNA.
Currently, in keeping with its traditional HIV focus, PEL is assisting Ad Bax's group at NIDDK in the search for potential structural partners that may help to stabilize the HIV nef protein. Meanwhile, on an exciting front that appears unrelated to HIV at first glance, PEL researchers are collaborating with NIAMS' Adam Zlotnick in efforts to delineate the apparently unique structure of a protein that coats the nucleus of the hepatitis B virus (HBV). The structure of most viral nucleocapsid proteins consists primarily of beta sheets arranged in a "jelly roll" formation. However, preliminary findings indicate that the HBV nucleocapsid protein, HBcAG, contains many alpha helixes arranged in a non-jelly roll fashion, Wingfield says. There are also intriguing early leads indicating that an HIV nucleocapsid protein, p27, may possess a structure similar to HBcAG's, he adds.
Intramural researchers who have come to rely on PEL for their HIV protein needs shouldn't lose any sleep worrying about the lab's impending move out of OD and into NIAMS, Wingfield says. In fact, he says the addition of a couple of postdocs could enhance PEL's ability to provide both proteins and protein-expression expertise to the NIH community. "I think this is an excellent move. It will make PEL a more settled part of the NIH family as a research group," says Steven.
Although the addition of PEL will definitely be a boon to the research capacity of a small and growing institute like NIAMS, Steven says he's certain that the lab "will continue to be polyvalent in terms of its scientific interactions, and will continue to collaborate on a campus-wide basis."
PEL's leaders also have a few nonadministrative changes up their scientific sleeves. The lab has recently extended its activities into protein crystallization in order to advance its interest in structure-function studies. "We feel if we can make the crystals ourselves, it will be more efficient than if we just hand the proteins out to people who may not have our understanding of the proteins," Wingfield says. "It's a routine part of our job to understand the properties and behaviors of the proteins we make."
With the arrival of its long-awaited postdocs, PEL may begin exploring new systems for the expression of proteins that undergo posttranslational modification. Although E. coli works well as a system in which large quantities of recombinant proteins can be produced, it lacks the factors necessary to perform the appropriate glycosylation, folding, and other posttranslational modifications required to make many proteins biologically active. One possible alternative may be a system in which genes are inserted via baculovirus and expressed in insect cells, Wingfield says.
Another problem that PEL is working on in collaboration with Norman Walts of NIAMS centers on the rather unruly behavior of some HIV proteins during handling and purification. Take the example of the HIV rev protein, which tends to polymerize into fibers that do not produce good mapping data under standard X-ray crystallography. Rather than fight rev's natural resistance to crystallization, PEL is now trying to chart rev's structure via X-ray diffraction of the polymerized fibers -- an innovative technique that has been used to plot the structure of the tobacco mosaic virus at high resolution.
Finally, Wingfield is also trying to wean the intramural research community away from simply viewing PEL as a one-way street that delivers much-needed proteins to their labs. Instead, he wants scientists to regard PEL as a two-way street for the exchange of information on protein expression at NIH. "Other researchers can help us by letting us know about their interesting ideas on protein expression or new insights on the structure or function of HIV proteins," he says.
PEL at a Glance
Paul Wingfield, lab chief, expert on protein structure-function.
Stephen Stahl, head of molecular biology section, expert on recombinant-DNA techniques and protein expression systems.
Location: Building 6B, Room 1B130.
Resources: This laboratory supplies HIV proteins that are not readily available from commercial suppliers to intramural scientists for collaborative studies. It also provides consultation and help on the expression and purification of recombinant proteins and assists investigators involved in such projects.