A model invalidation-based approach for elucidating biological signalling pathways,applied to the chemotaxis pathway in R. sphaeroides

Background  

Developing methods for understanding the connectivity of signalling pathways is a major challenge in biological research. For this purpose, mathematical models are routinely developed based on experimental observations, which also allow the prediction of the system behaviour under different experimental conditions. Often, however, the same experimental data can be represented by several competing network models.     

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.     

Mutations in the S6 Gate Isolate a Late Step in the Activation Pathway and Reduce 4-AP Sensitivity in Shaker Kv Channel

K_v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.     

CHARMM All-Atom Additive Force Field for Sphingomyelin: Elucidation of Hydrogen Bonding and of Positive Curvature

The C36 CHARMM lipid force field has been extended to include sphingolipids, via a combination of high-level quantum mechanical calculations on small molecule fragments, and validation by extensive molecular dynamics simulations on N-palmitoyl and N-stearoyl sphingomyelin. NMR data on these two molecules from several studies in bilayers and micelles played a strong role in the development and testing of the force field parameters. Most previous force fields for sphingomyelins were developed before the availability of the detailed NMR data and relied on x-ray diffraction of bilayers alone for the validation; these are shown to be too dense in the bilayer plane based on published chain order parameter data from simulations and experiments. The present simulations reveal O-H:::O-P intralipid hydrogen bonding occurs 99% of the time, and interlipid N-H:::O=C (26-29%, depending on the lipid) and N-H:::O-H (17–19%). The interlipid hydrogen bonds are long lived, showing decay times of 50 ns, and forming strings of lipids, and leading to reorientational correlation time of nearly 100 ns. The spontaneous radius of curvature for pure N-palmitoyl sphingomyelin bilayers is estimated to be 43–100 Å, depending on the assumptions made in assigning a bending constant; this unusual positive curvature for a two-tailed neutral lipid is likely associated with hydrogen bond networks involving the NH of the sphingosine group.