January 23, 2004
UCSD Researchers Develop New Way To Look At 3-D Protein Structures

Advances in basic instrumentation and in techniques for characterizing the structure of biological molecules enable many other advances to be made that produce results that are more directly usable in medicine and in other fields. While people who produce medical treatments tend to get most of the glory the scientists who make advances in instrumentation and in biological assays create the tools that make possible the many advances which are of more direct benefit.

With this thought in mind you might therefore be mildly excited to learn that some researchers at UC San Diego have developed a new method to determine the structure of proteins whose structures could not be determined by existing methods.

An innovative method that allows increased success and speed of protein crystallization – a crucial step in the laborious, often unsuccessful process to determine the 3-dimensional structure unique to each of the body’s tens of thousands of folded proteins – has been developed by researchers at the University of California, San Diego (UCSD) School of Medicine and verified in tests with the Joint Center for Structural Genomics (JCSG) at The Scripps Research Institute (TSRI) and the Genomics Institute of the Novartis Research Foundation in La Jolla, California.

Described in the Jan. 20, 2004 issue of the journal Proceedings of the National Academy of Sciences (PNAS)*, the method, which employs a UCSD invention called enhanced amide hydrogen/deuterium-exchange mass spectrometry, or DXMS, rapidly identifies small regions within proteins that interfere with their ability to crystallize, or form a compact, folded state. The investigators demonstrate that once these regions are removed by what amounts to “molecular surgery”, the proteins then crystallize very well.

“Although the sequencing of the human genome gave us the code for genes that are the recipes for proteins, we need to see and understand the folded shape taken by proteins to determine how they work as the fundamental components of all living cells,” said UCSD’s Virgil Woods, Jr., M.D., the inventor of DXMS, senior author of the PNAS article and an associate professor of medicine. “Definition of a protein folded structure is of great use in the discovery of disease-targeting drugs. Furthermore, when we’re able to identify incorrectly folded proteins in disease states, such as Alzheimer’s, cystic fibrosis and many cancers, we may then be able to design drugs that encourage proper folding or block the misshapen protein.”

The 3-dimensional structure of a protein is a useful piece of knowledge for drug developers. Detailed knowledge of a protein structure is a useful starting point to suggest what types of chemical compounds to build to test against a protein for binding affinity. Better protein structure determination tools will therefore speed drug development.

X-rays are used to gather information used to discover 3-dimensional structures of proteins. The problem is that x-ray crystallography requires that a protein first be induced to form crystalline structures and not all proteins can be made to do so.

Unfortunately, many proteins do not naturally form a single, compact state in solution and hence, they are often highly resistant to crystallization, which is required for the x-ray crystallographic process that determines their shape. X-ray crystallography works by bombarding x-rays off crystals of a protein that contain a 3-dimensional lattice, or array of the individual protein or of a protein complex. The scattered, or diffracted pattern of the x-ray beams is used to calculate a s-dimensional structure of the protein.

Out of 24 proteins used to test this technique, including 18 which existing techniques had failed to crystallize, the researchers were able to determine the structures of the 6 easy ones and 15 of the hard ones. Then with some genetic manipulation the researchers were able to determine the structures of 2 of the 3 remaining proteins.

Of the 24 proteins provided by JCSG for DXMS analysis, six had already been crystallized and their structures determined. The results provided by DXMS matched the information on those six proteins, correctly identifying even small unfolded regions. The remaining 18 proteins provided by JCSG had all failed extensive prior crystallization attempts. In the new experiments, DXMS technology rapidly determined the unstructured regions in 15 of these proteins.

Two of the previously failed proteins were then subjected to “molecular surgery”, in which the DXMS-identified unstructured regions were selectively removed from the DNA that coded for the proteins. DXMS study of the resulting modified proteins demonstrated that the surgery had removed the unstructured regions without otherwise altering the shape of the originally well-folded regions. Each of the two resulting DXMS stabilized forms of the proteins were then found to crystallize well, while the original, unmodified proteins again failed to crystallize.

JCSG investigators were subsequently able to determine the 3-dimensional structures of these two proteins by x-ray analysis of the crystals resulting from DXMS-guided stabilization. One of the proteins that was successfully crystallized was found to have a unique shape or “fold”, not previously seen in proteins.

So now a technique has been found that can determine the 3-dimensional structures of proteins which were previously beyond the reach of researchers. Science marches on.

Share |      Randall Parker, 2004 January 23 01:29 PM  Biotech Assay Tools

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