Category Archives: Biochemistry

Jan. 22, 2013 While working out the structure of a cell-killing protein produced by some strains of the bacterium Enterococcus faecalis, researchers stumbled on a bit of unusual biochemistry. They found that a single enzyme helps form distinctly different, three-dimensional ring structures in the protein, one of which had never been observed before.

The new findings, reported in Nature Chemical Biology, should help scientists find new ways to target the enterococcal cytolysin protein, a "virulence factor that is associated with acute infection in humans," said University of Illinois chemistry and Institute for Genomic Biology professor Wilfred van der Donk, who conducted the study with graduate student Weixin Tang.

Enterococcus faecalis (EN-ter-oh-cock-us faye-KAY-liss) is a normal microbial inhabitant of the gastrointestinal tracts of humans and other mammals and generally does not harm its host. Some virulent strains, however, produce cytolysin (sigh-toe-LIE-sin), a protein that, once assembled, attacks other microbes and kills mammalian cells.

"The cytolysin protein made by Enterococcus faecalis consists of two compounds that have no activity by themselves but when combined kill human cells," van der Donk said. "We know from epidemiological studies that if you are infected with a strain of E. faecalis that has the genes to make cytolysin, you have a significantly higher chance of dying from your infection." E. faecalis contributes to root canal infections, urinary tract infections, endocarditis, meningitis, bacteremia and other infections.

Enterococcal cytolysin belongs to a class of antibiotic proteins, called lantibiotics, which have two or more sulfur-containing ring structures. Scientists had been unable to determine the three-dimensional structure of this cytolysin because the bacterium produces it at very low concentrations. Another problem that has stymied researchers is that the two protein components of cytolysin tend to clump together when put in a lab dish.

Van der Donk and Tang got around these problems by producing the two cytolysin components separately in another bacterium, Escherichia coli (esh-uh-REE-kee-uh KOH-lie), and analyzing them separately.

"The two components are both cyclic peptides, one with three rings and the other with two rings," van der Donk said. "Curiously, a single enzyme makes both compounds."

In a series of experiments, the researchers found that one ring on each of the proteins adopted a (D-L) stereochemistry that is common in lantibiotics (see image, above). But the other rings all had an unusual (L-L) configuration, something van der Donk had never seen before.

Scientists had assumed that the enzyme that shaped enterococcal cytolysin, a lantibiotic synthetase, acted like a three-dimensional mold that gave the ring structures of cytolysin the exact same stereochemistry, van der Donk said.

"But we found that the enzyme, enterococcal cytolysin synthetase, makes the rings with different stereochemistry," he said. "I don't know of any other examples where one enzyme can make very similar products but with different stereochemistries."

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Odd biochemistry yields lethal bacterial protein

Newswise FAYETTEVILLE, Ark. After years of experimentation, researchers at the University of Arkansas have solved a complex, decades-old problem in membrane biochemistry. The consequence of their work will give scientists more information about the function and structure of proteins, the workhorses within the cells of the human body.

Historically, lysine and arginine, both basic amino acids, were considered to have very similar properties and therefore to be essentially interchangeable, said Denise Greathouse, a research associate professor in the department of chemistry and biochemistry. Our results demonstrate that despite their similarities, the differences in their behavior in membrane environments provide important clues for understanding membrane protein function.

The findings, which appear in the January issue of the journal Proceedings of the National Academy of Sciences, address long-standing questions in the study of protein structure and function and help explain how charged amino acids are able to modulate the behavior of proteins in cellular membranes.

Greathouse, former doctoral students Nicholas Gleason and Vitaly Vostrikov, and Roger Koeppe II, Distinguished Professor of chemistry and biochemistry, wrote the article, Buried lysine, but not arginine, titrates and alters transmembrane helix tilt.

Proteins do nearly all the work in the cells of our bodies, ranging from brain function and nerve transmission to metabolic energy production and muscular contraction. Moreover, many diseases are associated with defects in protein function. Future advances in the diagnosis and treatment of human disease will depend upon better understanding of the thousands of proteins that are encoded within the genomes of humans and human pathogens.

The structure and function of membrane proteins both play a crucial role in cell signaling and the regulation of biological function. The authors developed experimental methods that determine how lysine and arginine interact in the lipid bilayer membrane environment. In the last 10 years there have been computational predictions of the behavior of lysine and arginine in the membrane but not methods to test those predictions.

It is the first measurement of its type, its complexity makes it an elegant method, and it opens the door for other people to apply these methods on biologically important problems, Koeppe said. There is a lot of interest in trying to understand whats going on in these membranes, especially with protein molecules that carry particular electric charges. Unless we can understand it at the fundamental level, then we cant extrapolate it to the nervous system. Were trying to develop foundational knowledge that is needed to understand the nervous system.

Were excited about this study because it makes available knowledge that other researchers can use, he said. Those making the computer predictions can refine their methods and make better predictions because they know that they were able to reproduce some of our results.

Lysine and arginine are ionizable, which means they can have a positive electric charge. The research team created a framework for experimentation that uses magnetic resonance imaging to measure whether the groups remain charged or become uncharged as the acidity or the pH of the environment is changed. To make their procedure work, the scientists synthesized peptides, which are chemical compounds consisting of several or more linked amino acids. To enable the magnetic resonance experiments, some of the hydrogen atoms in the peptides were replaced with deuterium, a heavy isotope of hydrogen.

Weve spent about 15 years doing this, Koeppe said. We developed first- and second-generation families of model peptides, and we examine them in model lipid membranes in order to understand the properties of real cell membranes and real cell proteins. This is at a molecular level. We are not even up to the cell yet.

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Researchers Solve Complex Problem in Membrane Biochemistry Through Study of Amino Acids

Public release date: 22-Jan-2013 [ | E-mail | Share ]

Contact: Diana Yates diya@illinois.edu 217-333-5802 University of Illinois at Urbana-Champaign

CHAMPAIGN, Ill. While working out the structure of a cell-killing protein produced by some strains of the bacterium Enterococcus faecalis, researchers stumbled on a bit of unusual biochemistry. They found that a single enzyme helps form distinctly different, three-dimensional ring structures in the protein, one of which had never been observed before.

The new findings, reported in Nature Chemical Biology, should help scientists find new ways to target the enterococcal cytolysin protein, a "virulence factor that is associated with acute infection in humans," said University of Illinois chemistry and Institute for Genomic Biology professor Wilfred van der Donk, who conducted the study with graduate student Weixin Tang.

Enterococcus faecalis (EN-ter-oh-cock-us faye-KAY-liss) is a normal microbial inhabitant of the gastrointestinal tracts of humans and other mammals and generally does not harm its host. Some virulent strains, however, produce cytolysin (sigh-toe-LIE-sin), a protein that, once assembled, attacks other microbes and kills mammalian cells.

"The cytolysin protein made by Enterococcus faecalis consists of two compounds that have no activity by themselves but when combined kill human cells," van der Donk said. "We know from epidemiological studies that if you are infected with a strain of E. faecalis that has the genes to make cytolysin, you have a significantly higher chance of dying from your infection." E. faecalis contributes to root canal infections, urinary tract infections, endocarditis, meningitis, bacteremia and other infections.

Enterococcal cytolysin belongs to a class of antibiotic proteins, called lantibiotics, which have two or more sulfur-containing ring structures. Scientists had been unable to determine the three-dimensional structure of this cytolysin because the bacterium produces it at very low concentrations. Another problem that has stymied researchers is that the two protein components of cytolysin tend to clump together when put in a lab dish.

Van der Donk and Tang got around these problems by producing the two cytolysin components separately in another bacterium, Escherichia coli (esh-uh-REE-kee-uh KOH-lie), and analyzing them separately.

"The two components are both cyclic peptides, one with three rings and the other with two rings," van der Donk said. "Curiously, a single enzyme makes both compounds."

In a series of experiments, the researchers found that one ring on each of the proteins adopted a (D-L) stereochemistry that is common in lantibiotics (see image, above). But the other rings all had an unusual (L-L) configuration, something van der Donk had never seen before.

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Study: Odd biochemistry yields lethal bacterial protein

OMICS Group- Journal of Biochemistry Analytical Biochemistry -2161-1009-1-119
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OMICS Group-Biochemistry Physiology Open Access-2168-9652-1-103
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