November 9, 2004
UPTON, NY - Scientists from the U.S. Department of Energy's Brookhaven National Laboratory and Stony Brook University have determined the two-dimensional crystal structure of a membrane protein involved in the process by which the Escherichia coli (E. coli) bacteria infects a human. This protein structure is a first step to better understanding how an E. coli infection begins, which may lead to information on how to block it.
"E. coli is responsible for urinary tract infections, one of the most prevalent diseases in the U.S.," said Brookhaven biologist Huilin Li, the lead researcher on the study, described in the November 2, 2004, online edition of the Journal of Molecular Biology. "Between 50 and 80 percent of U.S. women will experience a urinary tract infection at least once during their lifetimes."
"In the first stage of the infection, E. coli binds tightly to human kidney cells, using an 'adhesive protein' secreted by the cells through a membrane protein 'channel.' Our structure of this protein channel helps show how secretion occurs, which may eventually lead us to determine how to stop E. coli from attaching to the human cell," said Li.
The protein channel, known as "PapC," is a member of the "chaperone/usher" family, channels that not only provide a pathway for certain substances to leave a cell but also participate in preparing the substance for secretion. In this case, PapC gathers the "parts" that make up the adhesive and then guides the assembled adhesive out of the cell.
Li and his colleagues found that PapC consists of two main structural elements, with each part containing one opening, or pore. Each pore is approximately two nanometers (billionths of a meter) in diameter, and the entire structure is 11 nanometers in length and seven nanometers wide.
While this structure might suggest that PapC uses both pores simultaneously, the researchers think that only one of the two pores may be in use at once. However, the twin pore configuration might be necessary to coordinate the assembly and secretion of the adhesive. This is consistent with other membrane proteins that perform similar functions.
"Our finding provides new insight into how the adhesive protein is assembled and secreted, but we need to know more about this process," said Li. "A greater understanding of this will aid in the study and treatment of urinary tract infections and other related diseases."
To determine the structure, the researchers grew a two-dimensional crystal of PapC - a sheet with a thickness of just one protein. To image individual proteins in the crystal, they used a technique called cryo-electron microscopy. In this method, the crystal is cooled to about minus 300 degrees Fahrenheit using liquid nitrogen and placed in an electron microscope. This device bombards the sample with high-energy electrons, which scatter off the atoms in the crystal. A lens inside the microscope focuses these electrons, forming a high-resolution image, which is recorded using film or a digital camera. The recorded images are analyzed by a computer, yielding the structure of the protein molecule.
This method produced a top-down image of the protein channel from an "untilted" sheet of crystals - that is, the electron beam hit the sheet head-on. To determine the channel's three-dimensional structure, Li and his group plan to perform additional high-resolution imaging experiments using the same crystal sheet, but tilting it to large angles. This will allow the electrons to scatter off and produce an image of the protein channel's other sides.
This research is a collaborative effort between researchers in Brookhaven Lab's Biology Department, Tianbo Liu of Brookhaven's Physics Department, and David Thanassi, a biologist in Stony Brook University's Department of Molecular Genetics and Microbiology. The research was funded by a Brookhaven Laboratory Directed Research and Development grant, the National Institutes of Health, and the Office of Biological and Environmental Research within the U.S. Department of Energy's Office of Science.
2004-240 | Media & Communications Office
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