Protein shapeshifter
[April 1, 2008]
--------------------------------------------------------------------------------

Volkman, using NMR to unfold received wisdom on proteins




NMR spectroscopy may have overturned a forty-year old tenet of biochemistry by showing that the immune system protein lymphotactin can fold and refold sometimes ten times per second into unrelated structural forms. Until now, it had been assumed that once folded into their active form all proteins would stay pretty much the same. The discovery may advance cancer and neurological diseases research.

Proteins are simply strings of amino acids, stitched together with instructions provided by genes, and then folded into their unique three-dimensional structures by the natural processes of solvent exclusion, hydrogen-bond formation and electrostatics. In the post-genomic era, identifying and understanding the functions of the tertiary structure of proteins has become one of the primary focuses of biomedical research.

But, what if the whole concept of protein folding hinged on a misconception? What if, instead of being set from the start, the shape and structure of proteins were flexible?

Researchers at the Medical College of Wisconsin in Milwaukee think they have evidence to support such a supposition. Their results suggest that the immune system protein, lymphotactin, can rapidly shift its shape - up to ten times a second - between two totally unrelated structures, each with a unique role in defending the body.

The team, Brian Volkman, Robbyn Tuinstra, and Francis Peterson, provides details of their findings in the journal Proceedings of the National Academy of Sciences, and suggests that they alter a fundamental concept of biochemistry that was first established in the 1960s. More importantly, than overturning the received wisdom, however, is that the work could lead to new insights into how other proteins can change form. Protein misfolding is thought to occur in many diseases such as Alzheimer's, Parkinson's, Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, the spongiform encephalopathies (Creutzfeldt-Jakob, mad cow disease, scrapie) and many type of cancer.

"We used a cryoprobe-equipped 600 MHz Bruker NMR spectrometer for the work," Volkman told SpectroscopyNOW, "We used NMR in three distinct aspects of the study. First, we used routine 3D NMR methods and isotope-filtered 3D NOESY measurements to solve the 3D structure of the alternative, dimeric lymphotactin conformation which adheres to the extracellular matrix," he explains. In an earlier study, the team had solved the NMR structure of lymphotactin in the conformation that resembles other members of the chemokine family. Secondly, they used 2D 15N exchange NMR to show directly that the two conformations are interconverting at a rate of around ten times per second.

"Thirdly, we performed what might be called an 'NMR co-precipitation assay', analogous to a co-immunoprecipitation or GST pulldown assay. In this experiment, we monitored the 2D NMR spectrum of a mixture of the two lymphotactin structures and showed that heparin (the cell-surface carbohydrate that binds lymphotactin and other chemokines to the endothelial wall) bound only to the novel, non-chemokine-like conformation. Because this heparin-lymphotactin complex is insoluble, the NMR signals for this structure vanished, leaving only the NMR spectrum of the other conformation, the one that attracts white blood cells."

The researchers also used fluorescence spectroscopy (using a PTI spectrofluorometer) to monitor the interconversion between the two conformations based on changes in the intensity and wavelength of the intrinsic tryptophan sidechain fluorescence emission.

"While our discovery raises more questions on the protein folding enigma, we hope it generates intensified research to learn the complex processes of these devastating diseases," explains team leader Volkman, associate professor of biochemistry at MCW.

The researchers found that human lymphotactin, a regulatory protein released by the immune system to attract and activate white blood cells, exists naturally in two distinct structures. The newly identified form has no similarity to any other known protein, say the researchers. They also showed that each form has a unique role, one attaches to the interior wall of the blood vessel, and the other reaches out to grab white blood cells. This implies that a conversion from one lymphotactin structure to the other is most probably essential to its activation, explains Volkman.

"Proteins often have multiple functional states that are closely related to a single structure," he says, "In its natural state, however, we found that lymphotactin adopts two equally populated but unrelated structures that rapidly change from one to the other."

Related links:

Proc Natl Acad Sci, 2008, in press
Volkman Page
Stanford's Folding at Home Project
Article by David Bradley

http://www.spectroscopynow.com/coi/c...&chId=5&page=1