Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

Selective labeling of α-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C. S., Manshouri, T., and Atassi, M. Z. (1987)J. Prot. Chem. 6, 455–461; Garcia-Borron, J. C., Bieber, A. L., and Martinez-Carrion, M. (1987)Biochemistry 26, 4295–4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict “flipping” across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity. The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide- or nitroxide-bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.     

‘So you are a mestiza’: Exploring the consequences of ethnic and racial clumping in the US academy

This paper intends to open a dialogue about the use of two specific labels in the US (i.e. ‘Chicanas’ and ‘Latinas’), with a special emphasis on how they are used in the US academy as both markers and identities. More specifically, this paper explores recent trends in the US academy by which women of Latin American descent are lumped together under the rubric Latinas, and in many cases assumed to be equivalent to Chicanas. Arguing that academia must learn to be more thoughtful when creating, defining and adopting categories, the article warns against recreating the very power dynamics we find in mainstream US society by way of these specific labels. Finally, ‘So you are a mestiza’ reminds its readers that, as a practice, social justice asks for a fundamental recognition of a culturally pluralist, democratic society. In this kind of society, histories must endure and multiple realities must be acknowledged.     

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG–Salt Aqueous Two‐Phase Systems for Rapid Screening Applications

Aqueous two‐phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high‐value proteins directly from unclarified cell culture supernatants. In this context, effective high‐throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore‐labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2‐carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (K_p) measured in polyethylene glycol (PEG) 3350–phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.     

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.     

Synthesis and Properties of Bacteriorhodopsin Analogs Containing Electron-Density Labels in the Chromophore Moiety

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the -ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.