(mentions CMT) Structural biology scores with protein snapshot

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Structural biology scores with protein snapshot

NMR methods applied to largest membrane protein to date

http://www.eurekalert.org/pub_releases/2009-06/vumc-sbs062509.php

In a landmark technical achievement, investigators in the Vanderbilt Center for

Structural Biology have used nuclear magnetic resonance (NMR) methods to

determine the structure of the largest membrane-spanning protein to date.

Although NMR methods are routinely used to " take molecular pictures " of small

proteins, large proteins – and particularly those that reside within the cell

membrane – have been reluctant to smile for the camera.

In the June 26 issue of Science, , Ph.D., professor of

Biochemistry, and colleagues report the NMR structure of the large bacterial

protein diacylglycerol kinase (DAGK), a complex of three subunits that each

cross the membrane three times (for a total of nine membrane spans).

The group's ability to determine the NMR structure of DAGK suggests that similar

methods can now be used to study the structures of other membrane proteins.

" We're taking the methods that we used for diacylglycerol kinase and applying

them to high value targets such as G protein-coupled receptors, " said.

G protein-coupled receptors – the largest family of cell signaling proteins –

are targets for about half of all pharmaceuticals. is collaborating with

other Vanderbilt investigators to tackle G protein-coupled receptor structure

using both NMR and a complementary structural approach, X-ray crystallography.

DAGK may be a therapeutic target for certain types of bacterial infections. It

is a virulence factor in the bacteria Streptococcus mutans, which causes tooth

decay.

selected DAGK as a model for studying membrane enzymes when he started

his own research lab 17 years ago. DAGK is the smallest known kinase (a protein

that adds chemical groups called phosphates onto other molecules), and it is not

similar to any other known proteins.

The DAGK structure, said, " confirmed that this is a really strange

kinase. " The enzyme has a porch-like structure, with a wide opening for its

substrate diacylglycerol and the active site at the top of the porch.

" The active site looks nothing like any other kinase active site – it's a unique

architecture, " said.

The researchers also performed exhaustive mutagenesis studies in which they

characterized mutations at each amino acid in DAGK and used the data to map the

active site of the enzyme onto the structure. They identified two sets of

mutations that resulted in non-functional DAGK. One set altered the active site

so that it no longer did its job, and the second set caused the protein to fold

incorrectly (misfolding).

said the team was surprised to find that nearly all of the mutations

that caused misfolding were in the active site. The expectation, he explained,

is that mutations in the active site would cause a loss of function but would

not usually affect protein folding, whereas key residues for folding would be

located elsewhere in the protein to underpin the scaffold for the active site.

" Our study shows that you can't make that assumption, " he said.

cautions that investigators cannot simply predict the impact of a

mutation based on it being located in the active site. The finding has

implications for personalized medicine, which aims to use the predicted impact

of disease-causing mutations to make therapy decisions.

" The therapeutic strategy for addressing catastrophic misfolding versus simple

loss of function may be very different, " said.

and his team, who got interested in protein folding because of their

work with DAGK, are now pursuing structural studies of misfolded membrane

proteins that cause diseases including peripheral neuropathy

(Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.

" For proteins that misfold because of mutations, we're using NMR tools to

understand exactly what the mutations do to the proteins in terms of structure

and stability, " said. " We believe that understanding will lead to

predictions about how to intervene and avoid misfolding. "

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Co-authors include Wade Van Horn, Ph.D., Hak-Jun Kim, Ph.D., Ellis,

Ph.D., Arina Hadziselimovic, Endah Sulistijo, Ph.D., Murthy Karra, Ph.D., and

Changlin Tian, Ph.D., at Vanderbilt and Sönnichsen, Ph.D., at Christian

Albrechts University in Kiel, Germany.

said the