Specification of complementary participatory methods for SEAMLESS-IF

Environmental Economics and Policy,

Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of G(i)- and G(q)-mediated signaling

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein α subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Giα. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Gα subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Gα subunits on cellular membranes

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins

Decyl­ammonium octa­noate

The title compound, C10H24N+·C8H15O2 −, forms a layered structure in which inter­molecular N+—H⋯O hydrogen bonds connect anions and cations, forming a two-dimensional network parallel to (010). The n-alkyl chains of the decyl­ammonium cations pack according to an ortho­rhom­bic ‘subcell’ with approximate dimensions 5.1 × 7.3 Å, and they are significantly distorted from planarity

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.Peer reviewe

Impact of physio-chemical spinning conditions on the mechanical properties of biomimetic spider silk fibers

Artificial spider silk has emerged as a biobased fiber that could replace some petroleum-based materials that are on the market today. Recent progress made it possible to produce the recombinant spider silk protein NT2RepCT at levels that would make the commercialization of fibers spun from this protein economically feasible. However, for most applications, the mechanical properties of the artificial silk fibers need to be improved. This could potentially be achieved by redesigning the spidroin, and/or by changing spinning conditions. Here, we show that several spinning parameters have a significant impact on the fibers' mechanical properties by tensile testing more than 1000 fibers produced under 92 different conditions. The most important factors that contribute to increasing the tensile strength are fast reeling speeds and/or employing post-spin stretching. Stretching in combination with optimized spinning conditions results in fibers with a strength of >250 MPa, which is the highest reported value for fibers spun using natively folded recombinant spidroins that polymerize in response to shear forces and lowered pH.The mechanical properties of spider silk are known to be dependent on spinning conditions. Here, the tensile behavior of over 1000 biomimetic spider silk fibers spun under 92 different conditions are tested, resulting in a yield strength of more than 250 MPa

The role of guidelines and the patient's life-style in GPs' management of hypercholesterolaemia

BACKGROUND: Recent Swedish and joint European guidelines on hyperlipidaemia stress the high coronary risk for patients with already established arterio-sclerotic disease (secondary prevention) or diabetes. For the remaining group, calculation of the ten-year risk for coronary events using the Framingham equation is suggested. There is evidence that use of and adherence to guidelines is incomplete and that tools for risk estimations are seldom used. Intuitive risk estimates are difficult and systematically biased. The purpose of the study was to examine how GPs use knowledge of guidelines in their decisions to recommend or not recommend a cholesterol-lowering drug and the reasons for their decisions. METHODS: Twenty GPs were exposed to six case vignettes presented on a computer. In the course of six screens, successively more information was added to the case. The doctors were instructed to think aloud while processing the cases (Think-Aloud Protocols) and finally to decide for or against drug treatment. After the six cases they were asked to describe how they usually reason when they meet patients with high cholesterol values (Free-Report Protocols). The two sets of protocols were coded for cause-effect relations that were supposed to reflect the doctors' knowledge of guidelines. The Think-Aloud Protocols were also searched for reasons for the decisions to prescribe or not to prescribe. RESULTS: According to the protocols, the GPs were well aware of the importance of previous coronary heart disease and diabetes in their decisions. On the other hand, only a few doctors mentioned other arterio-sclerotic diseases like stroke and peripheral artery disease as variables affecting their decisions. There were several instances when the doctors' decisions apparently deviated from their knowledge of the guidelines. The arguments for the decisions in these cases often concerned aspects of the patient's life-style like smoking or overweight- either as risk-increasing factors or as alternative strategies for intervention. CONCLUSIONS: Coding verbal protocols for knowledge and for decision arguments seems to be a valuable tool for increasing our understanding of how guidelines are used in the on treatment of hypercholesterolaemia. By analysing arguments for treatment decisions it was often possible to understand why departures from the guidelines were made. While the need for decision support is obvious, the current guidelines may be too simple in some respects

Tying up the Loose Ends : A Mathematically Knotted Protein

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a "knot" depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.Peer reviewe