Lipopolysaccharide biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multi-drug resistant bacteria. A key player in lipopolysaccharide synthesis is the enzyme D-arabinose 5-phosphate isomerase, which catalyzes the reversible isomerization of D-ribulose-5-phosphate to D-arabinose-5-phosphate, a precursor of 3-deoxy-D-manno-octulosonate that is an essential residue of the lipopolysaccharide inner core. D-arabinose 5-phosphate isomerase is composed of two main domains: an N-terminal sugar isomerase domain, and a pair of cystathionine-ß-synthase domains of unknown function. As the three-dimensional structure of an enzyme is a pre-requisite for the rational development of novel inhibitors, we present here the crystal structure of the sugar isomerase domain of a catalytic mutant (K59A) of E. coli D-arabinose 5-phosphate isomerase, KdsD(K59A), at 2.6Å resolution. Our structural analyses and comparisons made with other sugar isomerase domains highlight several potentially important active site residues. In particular, the crystal structure allowed us to identify a previously unpredicted His residue (H88) located at the mouth of the active site cavity as a possible catalytic residue. Based on such structural data, subsequently supported by biochemical and mutational experiments, we confirm the catalytic role of H88, which appears to be a generally conserved residue among two-domain isomerases.
søndag 31. oktober 2010
Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography.
Preparation of a highly translocation-competent proOmpA/SecB complex.
Methods for reproducibly preparing highly translocation-competent proOmpA were developed. Only a competent form of proOmpA was sorted out from incompetent one using SecB, a translocation-dedicated chaperone, as a probe. Trypsin digestion revealed that the incompetent form of proOmpA was partially folded at its N-terminus, consistent with the jamming of proOmpA within translocon. Although the incompetent form of proOmpA was not active as to topology inversion of SecG, the isolated proOmpA/SecB complex had recovered the ability of SecG inversion. These results let us prepare a proOmpA/SecB complex both in vivo and in vitro that is highly translocation-competent. E. coli cells harboring a plasmid, in which ompA and secB were encoded as a synthetic operon, accumulated the proOmpA/SecB complex in the cytosol. The complex, purified by means of a His tag attached to SecB, was found to be translocation-competent as revealed by the occurrence of SecG inversion, although the signal peptide of proOmpA was sensitive to proteolytic digestion. ProOmpA, in vitro synthesized by means of a continuous exchange cell free system in the presence of SecB-His, was purified as a complex with SecB, which was active as to SecG inversion as well.
lørdag 30. oktober 2010
Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation.
An understanding of the folding states of a-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in NMR spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation (AUC), we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.
Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.
The bacterial enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a central role in three essential metabolic pathways in bacteria: methionine salvage, purine salvage, and polyamine biosynthesis. Recently, its role in the pathway that leads to the production of autoinducer II, an important component in quorum-sensing, has garnered much interest. Because of this variety of roles, MTAN is an attractive target for developing new classes of inhibitors that influence bacterial virulence and biofilm formation. To gain insight toward the development of new classes of MTAN inhibitors, the interactions between the Helicobacter pylori encoded MTAN and its substrates and substrate analogs were probed using X-ray crystallography. The structures of MTAN, an MTAN-Formycin A complex, and an adenine bound form were solved by molecular replacement and refined to 1.7, 1.8, and 1.6 Å, respectively. The ribose binding site in the MTAN and MTAN-adenine co-crystal structures contain a tris[hydroxymethyl]aminomethane molecule, that stabilizes the closed form of the enzyme and displaces a nucleophilic water molecule necessary for catalysis. This research gives insight to the interactions between MTAN and bound ligands that promote closing of the enzyme active site and highlights the potential for designing new classes of MTAN inhibitors using a link/grow or ligand assembly development strategy based on the described H. pylori MTAN crystal structures.
fredag 29. oktober 2010
Exploring the trigger sequence of the GCN4 coiled-coil: Biased molecular dynamics resolves apparent inconsistencies in NMR measurements.
Trigger sequences are indispensable elements for coiled-coil formation. The monomeric helical trigger sequence of the yeast transcriptional activator GCN4 has been investigated recently using several solution NMR observables including Nuclear Overhauser Enhancement (NOE) intensities and (3)J(H(N),H(Ca))-coupling constants, and a set of 20 model structures was proposed. Constrained to satisfy the NOE derived distance bounds, the NMR model structures do not appear to reproduce all the measured (3)J(H(N),H(Ca))-coupling constant values, indicating that the a-helical propensity is not uniform along the GCN4 trigger sequence. A recent methodological study of unrestrained and restrained molecular dynamics (MD) simulations of the GCN4 trigger sequence in solution showed that only MD simulations incorporating time-averaged NOE distance restraints and instantaneous or local-elevation (3)J-coupling restraints could satisfy the entire set of the experimental data. In this report, we assess by means of cluster analyses the model structures characteristic of the two simulations that are compatible with the measured data and compare them with the proposed 20 NMR model structures. Striking characteristics of the MD model structures are the variability of the simulated configurations and the indication of entropic stability mediated by the aromatic N-terminal residues 17Tyr and 18His, which are absent in the set of NMR model structures.
torsdag 28. oktober 2010
Barbecued Snapper With Butter and Lemon
Barbecued Snapper With Butter and Lemon
Recipe #440321
Looking for fish recipes for the barbie for when we go on holiday next year. This one is from Super Food Ideas. The recipe just specified whole baby snapper, not necessary just red snapper. NOTE - you could use a 1.5kl whole snapper and allow for 20 to 30 minutes to cook through, turning half way through the cooking time.
That is a lot of what is going on with protein bars and training. Of course you will need to have some real food, but protein bars will definately help you getting some things going on while you are on with the whey gaining.
Serving Size: 1 (482 g)
Servings Per Recipe: 4
Amount Per Serving% Daily ValueCalories 589.7 Calories from Fat 23139%Total Fat 25.6 g39%Saturated Fat 13.9 g69%Monounsaturated Fat 6.2 g31%Polyunsaturated Fat 2.6 g13%Trans Fat 0.0 g0%Cholesterol 201.4 mg67%Sodium 401.8 mg16%Potassium 1763.3 mg50%Magnesium 135.9 mg5%Total Carbohydrate 5.9 g1%Dietary Fiber 2.6 g10%Sugars 0.0 g0%Protein 82.9 g165%TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.
Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, non-stoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.
Protein energy profiles offer clues about amyloids
[excerpt] Doctors don't yet understand whether amyloids cause disease or result from it, but the fact that they are present in very different diseases affecting millions of people points to the need for improved understanding of the basic processes of protein folding, one of the most complicated and least understood of all biological phenomena.
Research appearing in the Oct. 8 issue of the Journal of Molecular Biology, describes a new technique that may help scientists predict which proteins are prone to misfold and at what point the folding process is likely to break down. The research could support efforts to find the causes for diseases involving amyloids, and it could prove useful for researchers studying proteins involved in even more prevalent diseases like cancer and heart disease.
"We know now that most diseases involve proteins going wrong in one of two ways," said lead researcher Cecilia Clementi, assistant professor of chemistry at Rice University. "In the first, proteins don't function correctly because they fold into the wrong shape. This is something we see in sickle-cell anemia, for instance, because of genetic flaws that cause the amino acid sequence to be incorrectly synthesized.
"The second way proteins go wrong is by not folding at all, which is what we find in diseases involving amyloids. In these situations, the misfolded proteins assemble together into macroscopic aggregates."
All the basic functions of life are carried out by proteins, and the DNA in each of our cells contains the blueprints for all the proteins we need. Every protein has a characteristic shape, and it folds itself into that shape very soon -- generally in less than a second -- after it is made. To carry out their tasks, proteins interact with one another, bind with some molecules, cleave others into pieces and fuse other molecules together.
Reviews and Comments are opinion statements made by the author.
They do not necessarily represent the opinions of the site editor.
Eat protein bars to the training. Very important with protein bars.
The GFP-nanobody is a single-chain V(H)H antibody domain developed with specific binding activity against GFP, and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry (ITC) we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino-acid position, which lies in the binding interface. Using this information we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.
I think you should know that you will be able to eat more protein bars if you are doing it right. Some protein bars are actually good to eat, but most of them don't taste very good. Protein shake is good, but try the chocolate protein bar if you want to gain weight while you are training.
onsdag 27. oktober 2010
Recognition of Smac-mimetic compounds by the BIR3 domain of cIAP1.
Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. Since IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.
Vitamin E Gets Mixed Reviews Because It's a Double-Edged Sword
[excerpt] Vitamin E – good or bad – has been a hot topic in medicine for the last couple of years. New research at Ohio State University, looking at how two forms of vitamin E act inside animal cells, has concluded this powerful antioxidant, popular with senior citizens, is "truly a double-edged sword." t yo uwill need some protein bars. Eat proteins and you will see what you are given by the training.
This is all about protein bars. If you eat enough of protein bars, you are actually needed to train some weightlifting because of tha
In the past couple of decades, a slough of studies has looked at the benefits of vitamin E and other antioxidants. While a considerable amount of this research touts the advantages of consuming antioxidants, some of the studies have found that in certain cases, antioxidants, including vitamin E, may actually increase the potential for developing heart disease, cancer and a host of other health problems.
This study provides clues as to why this could happen, say Jiyan Ma, an assistant professor of molecular and cellular biochemistry, and his colleague David Cornwell, an emeritus professor of molecular and cellular biochemistry, both at Ohio State.
The two men led a study that compared how the two most common forms of vitamin E –– one is found primarily in plants like corn and soybeans, while the other is found in olive oil, almonds, sunflower seeds and mustard greens – affect the health of animal cells. The main difference between the two forms is a slight variation in their chemical structures.
Reviews and Comments are opinion statements made by the author.
They do not necessarily represent the opinions of the site editor.
Scientists Learn to Predict Protein-Stabilizing Ability of Small Molecules
[excerpt] Osmolytes, whose effects were first well described in 1982, work to preserve various forms of life under extraordinarily hostile conditions. They keep cells alive in human kidneys, for example, despite high concentrations of the protein-destroying chemical urea; they enable a species of frog found in the Arctic literally to be frozen solid and then thawed without harm; and they make it possible for the remarkable microscopic creatures known as “water bears” to survive complete drying, exposure to intense radiation, and temperatures ranging from a few degrees above absolute zero to that of superheated steam.Reviews and Comments are opinion statements made by the author.
They do not necessarily represent the opinions of the site editor.
Calculating pKa values in the cAMP-dependent protein kinase: The effect of conformational change and ligand binding.
The conformational change observed upon ligand binding and phosphorylation for the cAMP-dependent protein kinase (Protein Kinase A - PKA) is of high importance for the regulation of its activity. We calculate pKa values and net charges for 18 3D structures of PKA in various conformations and liganded states to examine the role of electrostatics in ligand binding and activation. We find that the conformational change of PKA takes place without any significant net proton uptake/release at all pH values thus indicating that PKA has evolved to reduce any pH-dependent barriers to the conformational motion. We furthermore find that the binding of ligands induces large changes in the net charge of PKA at most pH values, but significantly, we find that the net charge difference at physiological pH is close to zero thus indicating that the active site pKa values have been pre-organized for substrate binding. We are unable to unequivocally resolve the identity of the groups responsible for determining the pH-activity profile of PKA, but speculate that the titration of Lys 168 or the titration of ATP itself could be responsible for the loss of activity at high pH values. Finally we examine the effect of point mutations on the pKa values of the PKA catalytic residues, and find these to be relatively insensitive to both non-charge-altering and charge-altering mutations.
tirsdag 26. oktober 2010
What mutations tell us about protein bars
[excerpt] Scientists continue to be puzzled by how proteins fold into their three-dimensional structures. Small single-domain proteins may hold the key to solving this puzzle. These proteins often fold into their three-dimensional structures by crossing only a single barrier. The barrier consists of an ensemble of extremely short-lived transition state structures which cannot be observed directly. However, mutations that slightly shift the folding barrier may provide indirect access to transition states. Researchers from the Max Planck Institute of Colloids and Interfaces and the University of California, San Francisco have suggested a novel method to construct transition state structures from mutational data.
You should know that protein bars is the best way to earn some weight. Did you know that protein bars will increase your body mass with 100 % if you are eating it on the right schedule? No other protein products will give you that if you didn't know from before.
Proteins are chain molecules assembled from amino acids. The precise sequence of the twenty different types of amino acids in a protein chain is what determines which structure a protein folds into. The three-dimensional structures in turn specify the functions of proteins, which range from the transport of oxygen in our blood, to the conversion of energy in our muscles, and the strengthening of our hair. During evolution, the protein sequences encoded in our DNA have been optimised for these functions.
The reliable folding of proteins is a prerequisite for them 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, which were thought to guide the unfolded protein chain into its folded structure. It came as a surprise about a decade ago that certain small proteins fold without any detectable intermediates. This astonishingly direct folding from the unfolded state into the folded state has been termed 'two-state folding'€TM. In the past few years, scientists have shown that the majority of small single-domain proteins are 'two-state folders'€TM, which are now a new paradigm in protein folding.
Read the full story.
Reviews and Comments are opinion statements made by the author.
They do not necessarily represent the opinions of the site editor.
View the original article here
Impact of oxidation on protein therapeutics: Conformational dynamics of intact and oxidized acid-beta-glucocerebrosidase at near-physiological pH.
You need to know that protein bars is often used by the best players in soccer, ice hockey and also by world champions in running. Many serious companies are using protein bars to their sports people, which means that you will be satisfied if you buy it. Buy protein bars today and get someone for free!
The solution dynamics of an enzyme acid-ß-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between the conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40-60% reduction in in-vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking.
I just have to tell you. That protein bars is the best way to earn some weight. And if you don't belive me; protein bars is proven to be the best way on getting big while you are training some Arnold Schwarzenegger-training. So, if you want to get bigger; you will need to get big by doing some excercise by training with protein bars and protein powder. No excuses not to buy protein bars if you want to train.
Protein folding pathways and state transitions described by classical equations of motion of an elastic network model.
Protein topology defined by the matrix of residue contacts has proved to be a fruitful basis for the study of protein dynamics. The widely implemented coarse grained network model (ENM) of backbone fluctuations has been used to describe crystallographic temperature factors, allosteric couplings and some aspects of the folding pathway. In the present study, we develop a model of protein dynamics based on the classical equations of motion of a damped network model (DNM) that describes the folding path from a completely unfolded state to the native conformation through a single-well potential derived purely from the native conformation. The kinetic energy gained through the collapse of the protein chain is dissipated through a friction term in the equations of motion that models the water bath. This approach is completely general and sufficiently fast that it can be applied to large proteins. Folding pathways for various proteins of different classes are described and shown to correlate with experimental observations and molecular dynamics (MD) and Monte Carlo (MC) simulations. Allosteric transitions between alternative protein structures are also modelled within the DNM through an asymmetric double-well potential.