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31.
Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore. 相似文献
32.
Sota Yagi Satoshi AkanumaAkihiko Yamagishi 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):553-560
A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L—derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded—has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues. 相似文献
33.
34.
《Molecular membrane biology》2013,30(2):183-192
The characterization of fluorescelnphosphatidylethanolamlne (FPE) as a real-time Indicator of the electrostatic nature of a membrane surface is described. The conditions appropriate for the labelling of membranes and the implementation of FPE as a tool to monitor the interactions of various peptides with model membranes are outlined. It is shown that of the membrane-active peptides studied, Naja naja kaouthla cardiotoxin and pyrularia thionin bind to certain model membranes without insertion. Whereas the leader sequence of the nuclear encoded subunit IV of mammalian cytochrome c oxidase (E.C. 1.9.3.1), known as p-25, and melittin appear to bind and then partially insert into the membrane. It seems evident also that melittin does not adopt a fully transmembrane configuration. Melittin is known to promote membrane lysis and by employing a rapid-kinetic technique it is shown that the time-course of such lysis does not appear to correlate with peptide binding, but following binding a significant proportion of melittin must become inserted into the membrane before lysis appears to commence. 相似文献
35.
We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions. 相似文献
36.
The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 Å. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N,N-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 m NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions.The authors wish to thank Dr. F.S. Cohen for making available unpublished data and for helpful comments. This work was supported by National Institutes of Health grant GM 37396 and by the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Initiative (E.R.K.) 相似文献
37.
38.
利用静电场提高IVF小鼠胚胎发育率的研究 总被引:3,自引:0,他引:3
本研究首先用静电场处理小鼠2细胞期胚胎,通过观察其发育率筛选了最佳处理剂量(场强和时间),在此基础上探讨了不同细胞期的静电刺激对小鼠胚胎发育的影响。结果表明:用静电场处理小鼠2细胞期胚胎,能显著提高胚胎的发育能力,最佳处理剂量的囊胚率从42.4%提高到64.5%,囊胚孵化率从20.0%提高到44.8%,与对照组存在极显著差异(P<0.01)。不同细胞期胚胎对静电刺激的敏感性不同,其中1细胞(授精后6h)和2细胞期(授精后24h)胚胎对电场刺激较为敏感。 相似文献
39.
M. R. Gunner Emil Alexov Eduardo Torres Samir Lipovaca 《Journal of biological inorganic chemistry》1997,2(1):126-134
The importance of electrostatic effects in determining the free energy of redox reactions in proteins such as cytochromes
and iron-sulfur complexes is well established. Several theoretical techniques have been used to analyze how the protein and
its environment combine to produce the observed electrochemical midpoints. The free energy of changing the cofactor charge
is influenced by the distribution of charges and dipoles in the protein, solvent and ions surrounding the protein, and by
the redistribution of these charges and dipoles coupled to the reaction. An outline of a consistent view for calculating these
effects will be presented and compared with other theoretical models. Heme redox potentials in yeast cytochrome c and the cytochrome subunit of photosynthetic reaction centers will be calculated to show how these protein structures produce
the observed electrochemistry.
Received, accepted: 26 November 1996 相似文献
40.
1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C3+ > C2+ > C+.2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor.3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane.4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C3+ > C2+ > C+, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding.5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane.6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations.7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory. 相似文献