Ion channels and D-amino acids

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Monitoring ion channel conformations in membranes utilizing a novel dual fluorescence quenching approach

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics, and function of membrane-spanning channels. We have analyzed the localization of the functionally important tryptophan residues of the membrane-bound channel and non-channel conformations of gramicidin utilizing a novel dual fluorescence quenching approach [G.A. Caputo, E. London, Biochemistry 42 (2003) 3265-3274]. In this paper, we show for the first time that the dual quenching approach is applicable to multiple tryptophan containing functional ion channel peptides such as gramicidin. Importantly, dual quenching is found to be sensitive to the membrane-bound conformations of this important model ion channel

Membrane interfacial localization of aromatic amino acids and membrane protein function

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Modulation of fluorophore environment in host membranes of varying charge

The net electrical charge of the biological membrane represents an important parameter in the organization, dynamics and function of the membrane. In this paper, we have characterized the change in the microenvironment experienced by a membrane-bound fluorescent probe when the charge of the phospholipids constituting the host membrane is changed from zwitterionic to cationic with minimal change in the chemical structure of the host lipid. In particular, we have explored the difference in the microenvironment experienced by the fluorescent probe 2-(9-anthroyloxy)stearic acid (2-AS) in model membranes of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) which are otherwise chemically similar, using the wavelength-selective fluorescence approach and other fluorescence parameters. Our results show that the microenvironment experienced by a membrane probe such as 2-AS is different in POPC and EPOPC membranes, as reported by red edge excitation shift (REES) and other fluorescence parameters. The difference in environment encountered by the probe in the two cases could possibly be due to variation in hydration in the two membranes owing to different charges

Dynamics of a membrane-bound tryptophan analog in environments of varying hydration: a fluorescence approach

Tryptophan octyl ester (TOE) represents an important model for membrane-bound tryptophan residues. In this article, we have employed a combination of wavelength-selective fluorescence and time-resolved fluorescence spectroscopies to monitor the effect of varying degrees of hydration on the dynamics of TOE in reverse micellar environments formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Our results show that TOE exhibits red edge excitation shift (REES) and other wavelength-selective fluorescence effects when bound to reverse micelles of AOT. Fluorescence parameters such as intensity, emission maximum, anisotropy, and lifetime of TOE in reverse micelles of AOT depend on [water]/[surfactant] molar ratio (w ). These results are relevant and potentially useful for analyzing dynamics of proteins or peptides bound to membranes or membrane-mimetic media under conditions of changing hydration

Novel insights into protein structure and dynamics utilizing the red edge excitation shift approach

A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a corresponding shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which depends on the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES extremely useful since hydration plays a crucial modulatory role in the formation and maintenance of organized molecular assemblies such as folded proteins in aqueous solutions and biological membranes. The application of REES as a powerful tool to monitor the organization and dynamics of a variety of soluble, cytoskeletal, and membrane-bound proteins is discusse

Therapeutic mammoplasty: a “wise” oncoplastic choice—lessons from the largest single-center cohort from Asia

IntroductionThe majority of breast cancer patients from India usually present with advanced disease, limiting the scope of breast conservation surgery. Therapeutic mammoplasty (TM), an oncoplastic technique that permits larger excisions, is quite promising in such a scenario and well suited to breast cancer in medium-to-large-sized breasts with ptosis and in some cases of large or multifocal/multicentric tumors. Here, we describe our TM cohort of 205 (194 malignant and 11 benign) patients from 2012 to 2019 treated at a single surgeon center in India, the largest Asian dataset for TM.MethodsAll patients underwent treatment after careful discussions by a multidisciplinary tumor board and patient counseling. We report the clinicopathological profiles and surgical, oncological, cosmetic, and patient-related outcomes with different TM procedures.ResultsThe median age of breast cancer patients was 49 years; that of benign disease patients was 41 years. The breast cancer cohort underwent simple (n = 84), complex (n = 71), or extreme (n = 44) TM surgeries. All resection margins were analyzed through intra-operative frozen-section assessment with stringent rad-path analysis protocols. The margin positivity rate was found to be 1.4%. A majority of the cohort was observed to have pT1–pT2 tumors, and the median resection volume was 180 cc. Low post-operative complication rates and good-to-excellent cosmetic scores were observed. The median follow-up was 39 months. We observed 2.07% local and 5.7% distal recurrences, and disease-specific mortality was 3.1%. At median follow-up, the overall survival was observed to be 95.9%, and disease-free survival was found to be 92.2%. The patient-reported outcome measures (PROMs) showed good-to-excellent scores for all types of TMs across BREAST-Q domains.ConclusionWe conclude that in India, a country where women present with large and locally advanced tumors, TM safely expands the indications for breast conservation surgery. Our results show oncological and cosmetic outcomes at acceptable levels. Most importantly, PROM scores suggest improved overall wellbeing and better satisfaction with the quality of life. For patients with macromastia, this technique not only focuses on cancer but also improves self-image and reduces associated physical discomfort often overlooked by women in the Indian setting. The popularization of this procedure will enable Indian patients with breast cancer to receive the benefits of breast conservation

Depth-dependent solvent relaxation in reverse micelles: a fluorescence approach

We report here depth-dependent solvent relaxation effects in the reverse micellar assembly using the deeply embedded probe NBD-cholesterol, a fluorescent cholesterol analogue in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group is covalently attached to the flexible acyl chain of cholesterol. Because of its deeper location, the NBD group of NBD-cholesterol is capable of reporting solvation dynamics in the deeper regions of the organized molecular assembly in which it is incorporated. NBD-cholesterol exhibits red edge excitation shift (REES) when incorporated into reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane with varying [water]/[surfactant] molar ratio (w<SUB>o</SUB>). Interestingly, the extent of REES increases with increasing w<SUB>o</SUB> implying that the overall motional restriction experienced by the reorienting solvent molecules is increased with increasing hydration. This is in contrast to the behavior of interfacially localized probes. In addition, our results show that with increasing w<SUB>o</SUB>, the NBD group of NBD-cholesterol experiences increased polarity as evidenced by the decrease in fluorescence lifetime and other fluorescence parameters such as fluorescence intensity. NBD-cholesterol could prove to be a useful probe for monitoring depth-dependent dynamics in organized molecular assemblies

Spectrin organization and dynamics: new insights

Spectrin is the major constituent protein of the erythrocyte cytoskeleton which forms a filamentous network on the cytoplasmic face of the membrane by providing a scaffold for a variety of proteins. In this review, several aspects of spectrin organization are highlighted, particularly with respect to its ability to bind hydrophobic ligands and its interaction with membrane surfaces. The characteristic binding of the fluorescent hydrophobic probes Prodan and pyrene to spectrin, which allows an estimation of the polarity of the hydrophobic probe binding site, is illustrated. In addition, the contribution of uniquely localized and conserved tryptophan residues in the 'spectrin repeats' in these processes is discussed. A functional implication of the presence of hydrophobic binding sites in spectrin is its recently discovered chaperone-like activity. Interestingly, spectrin exhibits residual structural integrity even after denaturation which could be considered as a hallmark of cytoskeletal proteins. Future research could provide useful information about the possible role played by spectrin in cellular physiology in healthy and diseased states