May 28, 2007

2007 Week 21: Ferret Studies in Genetics


Someone was kind enough to post this to the Ferret Genetics group @Yahoo recently, so as a bonus, I decided to introduce them here also.

Mating system and genetic variance in a polygynous mustelid, the European polecat

The population genetic implications of mating system were investigated in European polecat Mustela putorius populations from western France, combining radiotracking survey and allozyme variation analysis. Mating peroid occurred from February to June and polecats showed a strategy of successive polygyny, a male consorting with 1.44 females during a brief period (2.9 days). Relatedness was largely sex biased, females (21%) being almost twice more related than males (13%) suggesting a natal philopatry. Nonetheless, breeding dispersal pattern appeared relatively complex. Males were the sex dispersing but the main strategy for male polecats consisted of short-term mating excursions in adjacent females ranges whereas long-distance dispersal only constituted an alternative breeding strategy. Despite their allozymic polymorphism level reaching 24% at p<0.05 for 38 scored loci, populations showed a high heterozygote deficiency as revealed by the FIS index averaging FIS=0.383. Thus the mating system of such solitary mustelids may be poorly efficient to prevent inbreeding within populations.

Genetic Heterozygosity in Polecat Mustela Putorius Populations from Western France

Allozymic variations were investigated in 49 European polecats Mustela putorius from Western France by starch gel electrophoresis. Out of 31 surveyed loci, eight (25.8%) were shown polymorphic and observed heterozygosity averaged 0.057. Deviations from Hardy–Weinberg equilibrium and heterozygote deficiency suggest that populations were not in panmixia. Heterozygotes for two loci or more totalled 42.9% of individuals. Thus, although carnivores were previously considered as less variable, polecat populations from Western France showed a high genetic variability.

Genetic variability in Danish polecats Mustela putoriusas assessed by microsatellites

Genetic variability and population structure was investigated in 83 European polecats Mustela putorius by means of six microsatellite markers. The samples came from two areas in Denmark, Østjylland and Thy, which are separated by the Limfjord. The genetic diversity (He = 0.583) found in the total sample was similar to those found in other mustelid species and carnivores in general. A heterozygote deficiency, probably due to a Wahlund effect, suggested a further substructuring of the Danish sample. Population genetic substructuring was investigated in three different ways: by means of the program STRUCTURE, Wright’s F-statistics and by an assignment test. All the tests indicate a subdivision of the sample into two distinct groups, which is concordant with the two sampling locations, with an average genetic divergence of FST = 0.126 and RST = 0.1692. The higher genetic diversity found in the Thy population (He = 0.578), as compared to the Østjylland population (He = 0.420), could be explained by assuming two ancient waves of colonisation of the Danish peninsula. Tests for recent bottlenecks were conducted, and the results suggest no evidence of neither population decline nor expansion. Our study is the first one in which microsatellite markers are used on polecat samples, and one locus (mv54) was found to be diagnostic in distinguishing between American mink Mustela vison and European polecat.

Genetic divergence without spatial isolation in polecat Mustela putorius populations

Understanding how genetic divergence could exist without spatial isolation is a fundamental issue in biology. Although carnivores have previously been considered as having a weak genetic variability, polecats Mustela putorius from eight distinct populations exhibited both a strong polymorphism (17.5-22.5%) and a substantial allele effective number reaching Ne=1.12. Heterozygosity ranging from Ho=0.031-0.063 significantly differed among populations, while the mean FIS averaging 0.388 stressed a real deficiency of heterozygotes. Observed heterozygosity levels among populations did not correlate with any habitat types but were clearly associated with habitat diversity index. The habitat structure in polecat home range corresponded to habitat mosaic structure in which discrete habitat types alternated causing multifactorial constraints that may favour heterozygosity. Allozymic frequencies within populations did not vary with dominant habitat. But in the Tyrosinase-1, the rare homozygote BB, resulting in a `dark' phenotype, was found much more in deciduous woods than the homozygote AA showing the `typical' pattern. Thus, the genetic basis for a character differentiation was here evidenced in a remarkable situation without spatial isolation. Further, the very low proportion of heterozygotes for this locus suggests a disruptive effect and supports the prediction of intermediate phenotypes being at a disadvantage. This heterozygote deficit may also result from an assortative mating intra phenotype (homogamy). The divergence in polecat phenotypes showed that genetic differentiation can be induced by subtle variations in environment, a situation that is likely to be frequent in most natural populations, and emphasized the adaptive nature of habitat preference.

Genetic structure of the European polecat (Mustela putorius) and its implication for conservation strategies


During the last century, the European polecat Mustela putorius populations in most of Europe declined and survived in fragmented patches, because of habitat alterations and direct persecution. To assess the genetic consequences of the demographic decline and to describe the spatial pattern of genetic diversity, 250 polecats sampled at seven localities from five European countries - Poland, Denmark (southern Denmark and northern Denmark), Spain, Belgium (eastern and western) and the Netherlands - were screened by means of nine microsatellite loci.

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May 20, 2007

2007 Week 20: Proteins


Columbia scientists determine 3-dimensional structure of cell's 'fuel gauge'
Researchers at Columbia University Medical Center have uncovered the complex structure of a protein that serves as a central energy gauge for cells, providing crucial details about the molecule necessary for developing useful new therapies for diabetes and possibly obesity. A paper published online today in the journal Science details this structure, helping to explain one of the cell's most basic and critical processes.

Electrons travel through proteins like urban commuters
For Duke University theoretical chemist David Beratan, the results of his 15 years of studying how electrons make their way through some important protein molecules can be summed up with an analogy: how do big city dwellers get from here to there?
In the Friday, Feb. 2, issue of the journal Science, Beratan and two co-authors use similar logic to describe their unified description of electron movements through certain "electron-transfer" proteins that lie at the heart of many processes essential for life. Such processes include harvesting light in photosynthesis in plant cells and generating energy in animal cells. "I think we have discovered the physical framework for thinking about all such protein electron-transfer chemistry," Beratan said. "Having this rule book in place will let scientists pose some hard but interesting questions about evolutionary pressures on protein structures.

Indel-based targeting of essential proteins in human pathogens that have close host orthologue(s): Discovery of selective inhibitors for Leishmania donovani elongation factor-1
We propose a novel strategy for selective targeting of essential pathogen proteins that contain sizable indels (insertions/deletions) in their sequences compared with their host orthologues. This approach has been tested on elongation factor-1[alpha] (EF-1[alpha]) from the protozoan pathogen Leishmania donovani. Leishmania EF-1[alpha] is 82% identical to the corresponding human orthologue, but possesses a 12 aminoacid sequence deletion compared with human EF-1[alpha]. We used this indel-differentiated region to design small molecules that selectively bind to leishmania EF-1[alpha] and not to the human protein. Three unrelated molecules were identified with the capacity to inhibit protein synthesis in leishmania by up to 75% while exhibiting no effect on human protein translation. These candidates may serve as prototypes for future development of antiprotozoan therapeutics. More generally, these findings provide a basis for a novel drug design platform. This platform targets essential pathogen proteins that are highly conserved across species, and consequently would not typically be considered to be conventional drug targets. We anticipate that such indel-directed targeting of essential proteins in microbial pathogens may help address the growing problem of antibiotic resistance.

Is glycine a surrogate for a D-amino acid in the collagen triple helix?
Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with , = –70°, 175°. A recent computational study suggested that replacing these glycine residues with d-alanine or d-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more d-alanine or d-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with d-alanine or d-serine greatly disfavors the formation of a triple helix. Host–guest studies also revealed that replacing a single glycine residue with d-alanine is more destabilizing than is its replacement with l-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a d-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, > 0°) are critical determinants for its beneficial substitution with a d-amino acid in a protein.

Protein-protein recognition and interaction hot spots in an antigen-antibody complex: Free energy decomposition identifies “efficient amino acids”
The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method was applied to the study of the protein-protein complex between a camelid single chain variable domain (cAb-Lys3) and hen egg white lysozyme (HEL), and between cAb-Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson-Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of "efficient amino acids," which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein-protein complex that could be targeted by small molecules inhibitors are discussed.

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2007 Week 20: Ferret Medical Studies


The effect of inflammation on Fos expression in the ferret trigeminal nucleus
We have previously carried out detailed characterization and identification of Fos expression within the trigeminal nucleus after tooth pulp stimulation in ferrets. The aim of this study was to determine the effect of pulpal inflammation on the excitability of central trigeminal neurons following tooth pulp stimulation. Adult ferrets were prepared under anesthesia to allow tooth pulp stimulation, recording from the digastric muscle, and intravenous injections at a subsequent experiment. In some animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. After 5 d, animals were re-anaethetized, and the teeth were stimulated at 10 times the threshold of the jaw-opening reflex. Stimulation of all tooth pulps induced ipsilateral Fos in the trigeminal subnuclei caudalis and oralis. All non-stimulated animals showed negligible Fos labeling, with no differences recorded between inflamed and non-inflamed groups. Following tooth pulp stimulation, Fos expression was greater in animals with inflamed teeth than in animals with non-inflamed teeth, with the greatest effect seen in the subnucleus caudalis. These results suggest that inflammation increases the number of trigeminal brainstem neurons activated by tooth pulp stimulation; this may be mediated by peripheral or central mechanisms.

Neuronal vacuolation in an adult ferret
The brain of a ferret showing abnormal neurologic signs was evaluated by histopathologic, histochemical, immunohistochemical, and ultrastructural examinations. Extensive neuronal vacuolation was observed. Since the brain was negative for protease-resistant protein prion (PrP'"), it was concluded that this was not a case of transmissible spongiform encephalopathy.

Bioelectric properties of chloride channels in human, pig, ferret, and mouse airway epithelia

In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia--from human, mouse, pig and ferret--grown at the air-liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na(+) transport, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl(-) transport, and cAMP-sensitive Cl(-) transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR (inh)-172, and CFTR(inh)-GlyH101, to block cAMP-mediated Cl(-) transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na(+) transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl(-) transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl(-) secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function.

High-throughput immunophenotyping of 43 ferret lymphomas using tissue microarray technology
To validate the use of the tissue microarray (TMA) method for immunophenotyping of ferret lymphomas, a TMA was constructed containing duplicate 1-mm cores sampled from 112 paraffin-embedded lymphoma tissue specimens obtained from 43 ferret lymphoma cases. Immunohistochemical (IHC) expression of CD3, CD79alpha, and Ki-67 (MIB-1) was determined by TMA and whole mount (WM) staining of each individual case for result comparison. There was a high correlation between CD79alpha and CD3 results comparing ferret TMA and WM sections (kappa statistic 0.71-0.73 for single-core TMA and 0.79-0.95 for duplicate-core TMA) and between continuous data from Ki-67 staining of ferret TMA sections and WM sections (concordance correlation coefficients 0.77 for single cores and 0.87 for duplicate cores). Subsequently, a panel of commercially available antibodies was applied to the TMA for the analysis of expression in ferret lymphomas. The results of this study confirmed previously published results suggesting specific cross-reactivity of the applied IHC markers (CD3, CD79alpha, Ki67) with ferret lymphoma tissue. Other IHC markers (CD45Ro, bcl2, bcl10, MUM1, CD30, vimentin) were also expressed in subsets of the included ferret lymphomas. Further studies are necessary to determine the usefulness of these markers for diagnostic and prognostic evaluation of ferret lymphomas. In conclusion, the TMA technology was useful for rapid and accurate analysis of protein expression in large archival cohorts of ferret lymphoma cases.

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May 14, 2007

2007 Week 19: Renal Disease


Amyloidosis in the black-footed ferret (Mustela nigripes)
MM Garner, JT Raymond, TD O'Brien, RW Nordhausen, and WC Russell
J Zoo Wildl Med, March 1, 2007; 38(1): 32-41.
This study describes clinical, histologic, immunohistochemical and electron microscopic features of amyloid A amyloidosis occurring in black-footed ferrets (Mustela nigripes) from eight U.S. zoological institutions. Ferrets had nonregenerative anemia, serum chemistries consistent with chronic renal disease, and proteinuria. Amyloid was present in a variety of tissues, but it was most severe in renal glomeruli and associated with tubular protein loss and emaciation. Congo red/potassium permanganate (KMnO4) and immunohistochemical stains revealed that the amyloid was of the AA type. Concurrent diseases and genetic predisposition were considered the most important contributing factors to development of amyloidosis. Analysis of the genetic tree did not reveal convincing evidence of a common ancestor in the affected ferrets, but a genetic predisposition is likely because all the captive black-footed ferrets are related.

A disorder marked by the deposition of amyloid in various organs and tissues of the body that may be associated with a chronic disease such as rheumatoid arthritis, tuberculosis, or multiple myeloma.

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May 11, 2007

2007 Week 19: Related Articles


Knots, Origami, & Water
"Knot" To Be Undone, Researchers Discover Unusual Protein Structure
Researchers funded by the National Institute of General Medical Sciences have determined the structure of a protein with a surprising feature in it: a knot. This is the first time a knot has been found in a protein from the most ancient type of single-celled organism, an archaebacterium, and one of only a few times a knot has been seen in any protein structure.
"It's a surprising and different structure," said NIGMS' John Norvell, Ph.D., director of the Protein Structure Initiative. Protein folding theory previously held that forming a knot was beyond the ability of a protein. Joachimiak suggests that the newly discovered knot may stabilize the amino acid subunits of the protein.

MIT Finds Most Complex Protein Knot Ever Seen
Knots are rare in proteins - less than 1 percent of all proteins have any knots, and most are fairly simple. The researchers analyzed 32,853 proteins, using a computational technique never before applied to proteins at this scale.
Of those that had knots, all were enzymes. Most had a simple three-crossing, or trefoil knot, a few had four crossings, and the most complicated, a five-crossing knot, was initially found in only one protein - ubiquitin hydrolase.
The complicated knot found in ubiquitin hydrolase may prevent it from getting sucked into the proteasome as it works, Mirny said. The researchers hypothesize that proteins with complex knots can't be pulled into the proteasome as easily, and the knots may make it harder for the protein to unfold, which is necessary for degradation.
The same knot is found in ubiquitin hydrolase in humans and in yeast, supporting the theory that there is a connection between the knot and the protein's function. This also seems to suggest that the knot has been "highly preserved throughout evolution," Virnau said.

Since their initial screening, the researchers have discovered five-crossing knots in two other proteins - a brain protein whose overexpression and mutations are linked with cancer and Parkinson's disease, and a protein involved in the HIV replication cycle. They have also found examples of proteins that are closely related and structurally similar except for the presence or absence of a knot.

Origami Helps Scientists Solve Problems
"Origami helps in the study of mathematics and science in many ways," says Martin Kruskal, a mathematician at Rutgers University, "Using origami anyone can become a scientific experimenter with no fuss." Kruskal found that origami is simpler to develop than most scientific theories and a lot easier to apply.

'DNA Origami': Caltech Scientist Creates New Method For Folding Strands Of Dna To Make Microscopic Structures
In a new development in nanotechnology, a researcher at the California Institute of Technology has devised a way of weaving DNA strands into any desired two-dimensional shape or figure, which he calls "DNA origami."
"The construction of custom DNA origami is so simple that the method should make it much easier for scientists from diverse fields to create and study the complex nanostructures they might want," Rothemund explains.
Although Rothemund has hitherto worked on two-dimensional shapes and structures, he says that 3-D assemblies should be no problem. In fact, researchers at other institutions are already using his method to attempt the building of 3-D cages. One biomedical application that Rothemund says could come of this particular effort is the construction of cages that would sequester enzymes until they were ready for use in turning other proteins on or off.

Unexpected Similarities Between Raindrops And Proteins
Raindrops and proteins seem to have a lot in common. This has been shown in a new study by scientists at Umeå University in Sweden. The principle behind the formation of raindrops is very similar to how proteins fold. This knowledge is vital to our understanding of neurodegenerative diseases like ALS.

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May 5, 2007

2007 week 18: Gentle Intro to Proteins & Folding


Some articles to help you get your feet wet...
Follow the links for the full article as usual.


Rensselaer Researchers Develop Approach That Predicts Protein Separation Behavior:
Applying math and computers to the drug discovery process, researchers at Rensselaer Polytechnic Institute have developed a method to predict protein separation behavior directly fromprotein structure. This new multi-scale protein modeling approach may reduce the time it takes to bring pharmaceuticals to market and mayhave significant implications for an array of biotechnology applications, including bioprocessing, drug discovery, and proteomics,the study of protein structure and function.
"Weintend to test the model against more complicated protein structures aspart of its further development," said Breneman. "The outcome of this work will yield fundamental information about the complex relationship between a protein's structural features and its chemical binding properties, and also aid in evaluating its potential biomedical applications."

Beyond Biology: Simple System Yields Custom-designed Proteins:
The diversity of nature may be enormous, but for Michael Hecht it is just a starting point.Hecht, a Princeton professor of chemistry, has invented a technique for making protein molecules from scratch, a long-sought advance that will allow scientists to design the most basic building blocks of all living things with a variety of shapes and compositions far greater than those available in nature.
Nearly all the internal workings of living things are built from proteins. While genes are the "blueprints" for organisms, proteins are the products built from those instructions. The molecules that transmit signals in the brain, carry oxygen in the blood and turn genes on and off are all proteins.
Scientists have long wanted to design their own proteins, but doing so has proved a major challenge. Proteins are strings of chemical units called amino acids and are often more than 100 amino acids long. When cells make them, these long chains fold spontaneously into complex three-dimensional shapes that fit like puzzle pieces with other molecules and give proteins their unique abilities. There are 20 different amino acids, so the number of possible combinations is enormous. However, the vast majority of these combinations are useless because they cannot fold into protein-like structures.
The advance reported by Hecht and colleagues involves a simple system for designing amino acid sequences that fold like natural proteins. First publishing the idea in 1993, Hecht realized that some amino acids were strongly "water-loving" while others were "oil-loving." The two types naturally separate from each other, with the oil-loving ones clustering in the protein core and water-loving ones forming the perimeter. He also saw that natural proteins with good structures tend to have certain repeating patterns of oil-loving and water-loving amino acids. For example, taking a string of water-loving units -- no matter which ones -- and inserting any oil-loving unit every three or four positions typically creates proteins that fold into bundles of helices.

Lasers Improve Scientists' Understanding Of Complex Proteins:
By shooting lasers at an RNA polymerase (RNAP) and a strand of DNA, scientists have learned a critical component of how a complex protein develops.
Using a system called fluorescence resonance energy transfer (FRET) on a single molecule, a researcher at the Lawrence Livermore National Laboratory’s Physical Biosciences Institute (PBI) in collaboration with UCLA scientists found that the procedure that regulates genes in a strand of DNA is a single process.
Earlier studies done with less precision resulted in scientists believing that the beginning and end phases of RNAP copying a DNA strand into RNA were two different processes. Using FRET, however, the recent study suggests that “there is no mechanistic difference between the start and finish,” said Ted Laurence of Livermore’s PBI. RNAP is the molecular machine that serves as a gene transcription tool. When it attaches to a strand of DNA, RNAP transcribes genes to RNA, which then is translated into a protein.

Livermore & NIH Scientists Create Technique To Examine Behavior Of Proteins At Single Molecule Level:
The work, presented in the Aug. 29 edition of Science, marks the first time protein-folding kinetics has been monitored on the single-molecule level. Proteins are long chains of amino acids. Like shoelaces, they loop about each other or fold in a variety of ways, and only one way allows the protein to function properly. Just as a knotted shoelace can be a problem, a misfolded protein can do serious damage. Many diseases, such as Alzheimer's, cystic fibrosis, mad cow disease and many cancers result from misfolded protein.

The Path To A Folded Protein, Long A Subject Of Debate, Appears In Many Cases To Be Long And Winding:
It's a long-simmering debate in the world of physical chemistry: Does the folding of proteins into biologically active shapes better resemble a luge run fast, linear and predictable or the more freeform trajectories of a ski slope? New research from the University of Pennsylvania offers the strongest evidence yet that proteins shimmy into their characteristic shapes not via a single, unyielding route but by paths as individualistic as those followed by skiers coursing from a mountain summit down to the base lodge. The new support for a more heterogeneous model of protein folding comes in a paper published today on the Web site of the Proceedings of the National Academy of Sciences.

"The traditional view has been that a protein passes through a series of fixed reactions to reach its folded state," said senior author Feng Gai, a Penn chemist. "Our work suggests quite strongly that folding is a far richer phenomenon. Like skiers, some proteins rocket down an energy gradient to their destination while others take their time, meandering indiscriminately."

Gai's work subtly shifts scientists' understanding of one possible remedy: molecular chaperones, promising compounds that "rescue" misfolded proteins and are believed capable of blocking the progression of neurodegenerative disease. Rather than giving sluggish proteins the oomph to finish folding, the Penn work indicates that chaperones may return misfolded proteins to an unfolded state so they can start all over again.

"In the skiing analogy, chaperones could be thought of as rescue helicopters that return wayward skiers to the summit so they can try to make their way down the mountain again," said Gai, an assistant professor of chemistry at Penn.

Untangling The Protein Folding Problem:
Protein folding research is "undergoing explosive growth," according to an editorial by Jay Winkler, Ph.D., and Harry Gray, Ph.D., both chemists at the California Institute of Technology and guest editors for the special issue. "Protein folding was once considered an almost intractable problem," write Winkler and Gray, but new efforts "are beginning to reveal the secrets of this prototypal spontaneous self-assembly process."

The special journal issue focuses on the chemical kinetics of the folding phenomenon -- the rate of change as the protein assumes its three-dimensional structure -- and includes a study on recent efforts to make "real time" observations. Folding happens very quickly, which makes it difficult to observe.

For some proteins, the change occurs in milliseconds (thousandths of a second); for others, it can be even faster. Despite recent progress toward understanding the mechanisms of protein folding, scientists still don't agree on exactly how it happens, as evidenced in the journal by the differing conclusions of several articles about the same protein.

Proteins are involved in many vital roles in humans, including metabolism, immunity and muscle movement. They are made up of amino acids, and it is the sequence of these amino acids that determines the eventual folded structures of the proteins, as well as the actual mechanism of the folding process.

Since a protein's structure is a key factor in how it functions in the body, the goal for researchers is to be able to predict the final three-dimensional structure based on the amino acid sequence.

How Proteins Fold Into Their Critical Shapes:
Experimental evidence provided by a Cornell researcher and colleagues at the Scripps Research Institute in La Jolla, Calif., support a long-held theory of how and where proteins fold to create their characteristic shapes and biological functions.
The theory proposes that proteins start to fold in specific places along an amino acid chain (called a polypeptide chain) that contains nonpolar groups, or groups of molecules without a charge, and continue to fold by aggregation, i.e., as several individuals of these nonpolar groupings combine. Using the same principle that separates oil and water, these molecules are hydrophobic -- they avoid water and associate with each other.

In the water-based cell fluid, where long polypeptide chains are manufactured and released by ribosomes, the polypeptide chains rapidly fold up into their biologically functional structure. The theory proposes that there are sites along the polypeptide chains where hydrophobic groups initially fold in on themselves, creating small nonpolar (hydrophobic) pockets that are protected from the water.

Research Answers Key Question In Biochemistry: How Proteins Fold Into 3-D Structures:
In research published in the July 29 issue of Nature, U of T post-doctoral fellow Dmitry Korzhnev and his supervisor, Professor Lewis Kay of the Department of Biochemistry, become the first researchers to characterize at an atomic level of detail the intermediate -- or substructure -- that forms as a protein folds to its 3-D state.

"Understanding how proteins fold is one of the Holy Grails of biochemistry," says Kay. "The intermediates that we can study make up only one or two per cent of the population of protein molecules in solution. It's hard to study them because they are present at such low levels. This is the first time we have been able to characterize an intermediate state at this level of detail."

If scientists can understand the pathway a protein takes from one state to another, they may be able to predict protein structure, something that can't be done very reliably at present. The ability to accurately predict protein structure has implications for drug design, as well as for improving commercial products.

Understanding the pathway a protein follows will also help scientists understand errors in folding, a problem linked to diseases such as cystic fibrosis and Alzheimer's.

Most Stable Parts Of Protein Are The First To Fold, Study Finds:
Like a 1950's Detroit automaker, it appears that nature prefers to build its proteins around a solid, sturdy chassis.

A new study combining advanced computational modeling and cutting-edge experiments by molecular biologists at Rice University and Baylor College of Medicine suggests that the most stable parts of a protein are also the parts that fold first.

Nature refuses to choose between form and function when it comes to protein folding; each protein's function is directly related to its shape, and when proteins misfold -- something that's known to occur in a number of diseases like Alzheimer's and Huntington's -- they don't function as they should.

In the new study, scientists designed and tested a new computational approach that aimed to study proteins with known shapes in order to ascertain which of their parts were the most stable in the face of chemical and thermal fluctuations.

"As far as we know, no one has ever used this type of knowledge-based, statistical approach to predict the stability cores of proteins," Ma said. "Our results suggest that thermodynamics and kinetics are closely correlated in proteins and appear to have co-evolved for optimizing both the folding rate and the stability of proteins."

Quantum Leap In Protein Folding Calculations:
Applying techniques derived from classical and quantum physics calculations may radically reduce the time it takes to simulate the way that proteins fold.

It's vital to understand the shapes that proteins take on as they fold up because the shapes determine how they function, both in keeping cells running and in leading to various diseases.

Rather than calculating the motions of a protein molecule step by step, as most simulations do, a team of Italian and French physicists studied the evolution of a molecule using variational principles. The technique allowed the physicists to evaluate all the possible paths that the molecule's parts would follow and then pick out the most likely one.

As a result, they expect to streamline protein folding calculations from trillions of steps to hundreds. The improvement is significant because conventional protein folding simulations that currently require supercomputers or large PC farms could instead be solved with individual desktop PCs running variational principle calculations.

Comprehensive Model Is First To Map Protein Folding At Atomic Level:
Scientists at Harvard University have developed a computer model that, for the first time, can fully map and predict how small proteins fold into three-dimensional, biologically active shapes. The work could help researchers better understand the abnormal protein aggregation underlying some devastating diseases, as well as how natural proteins evolved and how proteins recognize correct biochemical partners within living cells.

The technique, which can track protein folding for some 10 microseconds -- about as long as some proteins take to assume their biologically stable configuration, and at least a thousand times longer than previous methods -- is described this week in the Proceedings of the National Academy of Sciences.

"For years, a sizable army of scientists has been working toward better understanding how proteins fold," says co-author Eugene I. Shakhnovich, professor of chemistry and chemical biology in Harvard's Faculty of Arts and Sciences. "One of the great problems in science has been deciphering how amino acid sequence -- a protein's primary structure -- also determines its three-dimensional structure, and through that its biological function. Our paper provides a first solution to the folding problem, for small proteins, at an atomic level of detail."

Fiendishly intricate, protein folding is crucial to the chemistry of life. Each of the body's 20 amino acids, the building blocks of proteins, is attracted or repulsed by water; it's largely these affinities that drive the contorting of proteins into distinctive three-dimensional shapes within the watery confines of a cell. The split-second folding of gangly protein chains into tight three-dimensional shapes has broad implications for the growing number of disorders believed to result from misfolded proteins or parts of proteins, most notably neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.

The model developed by Shakhnovich and colleagues faithfully describes and catalogs countless interactions between the individual atoms that comprise proteins. In so doing, it essentially predicts, given a string of amino acids, how the resulting protein will fold -- the first computer model to fully replicate folding of a protein as happens in nature. In more than 4,000 simulations conducted by the researchers, the computer model consistently predicted folded structures nearly identical to those that have been observed experimentally.


What Mutations Tell Us About Protein Folding:
Scientists continue to be puzzled by how proteins fold intotheir three-dimensional structures. Small single-domain proteins mayhold the key to solving this puzzle. These proteins often fold intotheir three-dimensional structures by crossing only a single barrier.The barrier consists of an ensemble of extremely short-lived transitionstate structures which cannot be observed directly. However, mutations that slightly shift the folding barrier may provide indirect access to transition states.

The reliable folding of proteins is a prerequisite forthem to function robustly. Mis-folding can lead to protein aggregates that cause severe diseases, such as Alzheimer's, Parkinson's, or the variant Creutzfeldt-Jakob disease. To understand protein folding,research has long focused on metastable folding intermediates, whichwere thought to guide the unfolded protein chain into its foldedstructure. It came as a surprise about a decade ago that certain smallproteins fold without any detectable intermediates. This astonishingly direct folding from the unfolded state into the folded state has been termed 'two-state folding'. In the past few years, scientists have shown that the majority of small single-domain proteins are 'two-state folders', which are now a new paradigm in protein folding.

The characteristic event of two-state folding is the crossing of a barrierbetween the unfolded and folded state. This folding barrier is thought to consist of a large number of extremely short-lived transition state structures. Each of these structures is partially folded and will either complete the folding process, or will unfold again, with equal probability. Transition state structures are thus similar to a ball ona saddle point, which has the same probability, 0.5, of rolling toeither side of the saddle.

Since transition state structures arehighly instable, they cannot be observed directly. To explore two-statefolding, experimentalists instead create mutants of a protein. Themutants typically differ from the original protein -- the wild type --in just a single amino acid. The majority of these mutants still foldinto the same structure, however the mutations may slightly change thetransition state barrier and, thus the folding time; that is, the timean unfolding protein chain on average needs to cross the foldingbarrier.

The central question is: can we reconstruct the transition state from the observed changes in the folding times? Such a reconstruction clearly requires experimental data on a large number of mutants. In the traditional interpretation, the structural information is extracted for each mutation, independent of the other mutations. Ifa mutation does not change the folding time, then the mutated amino acid traditionally is interpreted to be still unstructured in the transition state. In contrast, if a mutation changes the folding time,the mutated amino acid is interpreted to be partially or fully structured in the transition state, depending on the magnitude of the change.

In a recent article in PNAS, a research team from the MaxPlanck Institute of Colloids and Interfaces and the University of California, San Francisco has suggested a novel interpretation of the mutational data. Instead of considering each mutation on its own, the new interpretation collectively considers all mutations within acooperative substructure, such as a helix. In case of the ±-helix ofthe protein CI2, this leads to a structurally consistent picture, inwhich the helix is fully formed in the transition state, but has not yet formed significant interactions with the ²-sheet.

In the future, the Max Planck researchers hope to construct complete transition states from mutational data. An important step is to identify the cooperative subunits of proteins, which requires molecular modelling. In a similar way to how a mountain pass shows us how to cross the landscape, the transition states eventually may help us to understand how proteins navigate from the unfolded into the folded structure.

Penn Scientists Show How Mistakes In Protein Folding Are Caught By "Protein Cages" Called Chaperonins:
It's imperative for all biological processes that proteins correctly maneuver from a simple string of amino acids to their pre-destined three-dimensional structure. This transformation -- called protein folding - is one of the most active areas of molecular biological research, and has taken on even more importance with the growing knowledge that misfolding can lead to such disorders as Alzheimer's disease, Huntington's disease, and prion-related neurodegenerative diseases. Recently, researchers at the University of Pennsylvania Medical Center have discovered how proteins called chaperonins protect cells from harm by sequestering and unfolding misshapened proteins. A report on this study appears in the April 30 issue of Science.

"Proteins should know how to fold by themselves, but they sometimes get into trouble," says senior author S. Walter Englander, PhD, a professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine. In times of stress, cells produce chaperonins, which are huge protein molecules that police other proteins that have misfolded as a result of any number of stressors -- including heat, heavy-metal poisoning, and ultraviolet radiation. If left unchecked, the misfolded proteins tend to clump, which can be harmful to normal cellular functions.

The Penn study looked specifically at a chaperonin called GroEL. "GroEL grabs the misfolded protein, engulfs it, pulls it open, and then throws it back out into the cytoplasm of the cell to fend for itself, " explains Englander. "The protein then takes its chances -- it may fold successfully, or it may get into trouble again." This entire process takes place within 13 seconds.

GroEL -- a sandwich of two circular proteins with a large central core into which average-sized proteins can fit - is able to capture thousands of different types of misfolded proteins. Its cavity is ringed with sites that bind nonspecifically to the hydrophobic, or water-avoiding, portions of proteins, which are normally found tucked deep inside a properly folded protein. "When a protein is misfolded and its hydrophobic insides are exposed, chaperonins snatch them up and help them to fold correctly by forcing them to unfold, so that they can try again," notes Englander.

Mechanism Of Protein Folding Unraveled, With Eventual Implications For Treating Diseases Caused By Folding Errors:
How a protein manages to fold is a seemingly impossible problem, suggests S. Walter Englander, PhD, a professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine: "Even with a small, 100-amino-acid-long protein, the number of possible three-dimensional structures that the protein might manifest is larger than the number of molecules in the universe." Protein biologists believe that the amino-acid sequences laid out by the genetic machinery contain chemical instructions for the pathway that carries each protein to its final structure.

The Penn experiments show that the amino-acid chain progresses through a series of pre-determined, intermediate arrangements. Englander's lab has demonstrated that the protein cytochrome c builds its structure in steps by first making helices at either end that lay at right angles to each other. Then, strands, loops, and other helices build up against that initial foundation until the final arrangement is reached. All this can occur in less than one second, but trouble can arise along the way. "A few years ago we showed that on the complicated journey to their final structure, proteins have a large tendency to make mistakes that greatly slow them down," notes Englander. "Proteins need to fold fast because if they spend too much time in one intermediate state, they're vulnerable to aggregation with other proteins in the midst of folding, which can be very destructive to the cell."

The recent work straightens out misinterpretations about how fast this process can proceed. "Numerous papers published in the past two years all conclude that when you initiate folding in a rapid reaction experiment, you see some very fast sub-millisecond optical signal changes, as well as some slower ones" explains Englander. "This has always been interpreted as a rapid formation of some real structural intermediates. It is crucially important to understand which of these signals represent real protein behavior and which give you misleading clues that simply depend on the kind of experiment you are doing. Understanding the folding process and the real time scale of events begins to give you some idea of what you can do to fight diseases like Alzheimer's."

Englander's lab performed experiments that convincingly showed that these exceedingly fast initial signals are not real intermediates, but simply represent the protein stretching and pulling in the denaturing solution used in the experiment. The researchers made two copies of the same amino-acid chain, one that couldn't fold and one that could, and observed that both versions displayed the same initial ultra-fast burst of optical activity.

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