Researchers are developing a new hybrid method for studying the HIV protein involved in the progression of the disease -
More than 36 million people worldwide, including 1.2 million in the United States living with HIV. antiretroviral cocktails block today how HIV replicates, matures and enters the uninfected cells, but they can not eradicate the virus.
Mike Kent, researcher in bioengineering and Sandia National Laboratories Science Centre, is studying a protein called Nef implicated in the progression from HIV to AIDS with the ultimate goal of the block. He and his collaborators have developed a new hybrid method for studying the HIV protein that compromises the immune system. The method could also work on many other proteins that damage cellular processes and cause disease.
Nef goes to the membrane of the infected cell and tricks the cell into destroying its own signaling system immune receptors, allowing the infected cell to evade the immune system. Nef also diverts cellular communications to make it easier for the virus to reproduce. To interact with host proteins, Nef needs to change shape.
This shape-shifting protein is so important that rhesus monkeys infected with a version of simian immunodeficiency virus closely related that lacks the protein Nef don 't develop symptoms of immune deficiency.
"Nef is an essential protein for AIDS. It accomplishes its tasks by modifying the signaling and receptor trafficking. It binds to receptors of the immune system critical and then signals your cells to destroy them. If you how this protein works, you have a better chance to develop drugs to stop it, "said Kent.
combining both techniques reveals the Nave structure and function
Kent and chemistry professor John Engen bioanalytical team at Northeastern University has combined two biophysical techniques known to find out how Nef alters the structure to perform its functions.
Kent is an expert in neutron reflectometry, a technique that gets the nanoscale structural information on films and biological membranes. his team used this technique to compare the Nave overall structure in form membrane bound from its inactive form, without membrane.
strong point Engen is hydrogen-deuterium exchange mass spectrometry, a technique that measures the local structure and protein flexibility. The team used to obtain information on the local structure and dynamics of Nef when bound to the membrane.
The comprehensive information of the neutron reflectometry shows that the average position of the Nave from the membrane. The local dynamics of hydrogen-deuterium exchange mass spectrometry vest for many small parts of the protein, showing the flexibility of 30 overlapping sections, which collectively cover 0 percent of Nef. Together they build a more complete picture of the Nave and structural changes.
The global and local information specific peptide supported a widespread assumption that, in binding to the membrane, Nef modifies its structure to interact with signaling receptors and other host proteins : .. a hypothesis unsupported, so far
"people have long studied Nef and there was a model of what the people thought that the protein might look like and could be a difficult protein NEF to study because you can crystallize the folded portion of the protein, and about half of the protein is unstructured. in addition, you can not study the membrane bound form crystallography, "said Kent.
"It is the first time anyone had measured these types of structural changes and the results were consistent with the hypothetical model," added Kent. "The details of these shape changes provide important new molecular knowledge of how NEF functions." This method could lead to new drug screening tests.
To combine the two techniques, the first team needed to make a special device. It should contain a lipid monolayer flat, saturated fat, which mimicked the biological membrane. It also had to be integrated with equipment for neutron sources for measuring the reflection of neutrons, and allowing the rapid exchange of the liquid carrier layer for the experiments hydrogen-deuterium exchange.
Another challenge was successfully produced the Nef protein. In infected cells, Nef is marked with a special lipid that serves to anchor Nef to the cell membrane. Engen's team had to produce Nef which contained this vital lipid, known as a group of myristate.
This work was supported by National Institutes of Health. Neutron reflection measurements were performed neutron research center at the National Institute of Standards and Technology and the Spallation Neutron Source at Oak Ridge National Laboratory.
New method could answer many questions about HIV, other. diseases
With the hybrid method and a unique camera in hand, the team is seeking funds to answer additional questions about Nef
"We studied alone, now we want explore with its binding partners, with host proteins and complexes that form in the presence of drug molecules or inhibitors, "said Kent. "Stop bind with partners or inhibiting to adopt the conformation that leads to the degradation of the receptor have important medical implications."
Tom Smithgall of the University of Pittsburgh School of Medicine, a co-author on a paper of the team, is currently the selection of potential drugs that could block the actions of Nef
Kent also hopes to apply this hybrid method to other important structural problems of proteins associated with the membrane, including virus maturation. the fusion of the virus with the membrane of the host cell; the functioning of bacterial toxins such as botulinum, tetanus and diphtheria; and dysfunctions ranging from cancer to the regulation of signaling cells cholesterol.
"There is a lot of potential for the combination of these two techniques in a more general sense. There is no other way to get this kind of specific, direct information on membrane proteins essential. This is an important segment of biological problems that could not be tackled in our work, and we made some big steps forward. future earnings depends on the general, we can apply the method beyond celui- HIV proteins, "said Kent.