ALBERT

Description: 〈p〉Publication date: Available online 5 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Thomas M. Kennelly, Yiran Li, Yi Cao, Eva E. Qwarnstrom, Mark Geoghegan〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Dynamic single-molecule force spectroscopy was performed to monitor the unbinding of fibronectin with the proteoglycans syndecan-4 (SDC4) and decorin and to compare this with the unbinding characteristics of 〈em〉α〈/em〉〈sub〉5〈/sub〉〈em〉β〈/em〉〈sub〉1〈/sub〉-integrin. A single energy barrier was sufficient to describe the unbinding of both SDC4 and decorin from fibronectin, whereas two barriers were observed for the dissociation of 〈em〉α〈/em〉〈sub〉5〈/sub〉〈em〉β〈/em〉〈sub〉1〈/sub〉-integrin from fibronectin. The outer (high-affinity) barriers in the interactions of fibronectin with 〈em〉α〈/em〉〈sub〉5〈/sub〉〈em〉β〈/em〉〈sub〉1〈/sub〉-integrin and SDC4 are characterized by larger barrier heights and widths and slower dissociation rates than those of the inner (low-affinity) barriers in the interactions of fibronectin with 〈em〉α〈/em〉〈sub〉5〈/sub〉〈em〉β〈/em〉〈sub〉1〈/sub〉-integrin and decorin. These results indicate that SDC4 and (ultimately) 〈em〉α〈/em〉〈sub〉5〈/sub〉〈em〉β〈/em〉〈sub〉1〈/sub〉-integrin have the ability to withstand deformation in their interactions with fibronectin, whereas the decorin-fibronectin interaction is considerably more brittle.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 3 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Sabine Oldemeyer, Maria Mittag, Tilman Kottke〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga 〈em〉Chlamydomonas reinhardtii〈/em〉 has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark. Concomitant with photoconversion of the flavin, an unusually long-lived tyrosyl radical with a red-shifted ultraviolet-visible spectrum is formed, which is essential for the function of the receptor. In this study, the microenvironment of this key residue, tyrosine 373, was scrutinized using time-resolved Fourier transform infrared spectroscopy on several variants of animal-like cryptochrome and density functional theory for band assignment. The reduced tyrosine takes on distinct hydrogen bond scenarios depending on the presence of the C-terminal extension and of a neighboring cysteine. Upon radical formation, all variants showed a signal at 1400 cm〈sup〉−1〈/sup〉, which we assigned to the 〈em〉ν〈/em〉7′a marker band of the CO stretching mode. The exceptionally strong downshift of this band cannot be attributed to a loss of hydrogen bonding only. Time-resolved ultraviolet-visible spectroscopy on W322F, a mutant of the neighboring tryptophan residue, revealed a decrease of the tyrosyl radical lifetime by almost two orders of magnitude, along with a shift of the absorbance maximum from 416 to 398 nm. These findings strongly support the concept of a 〈em〉π〈/em〉-〈em〉π〈/em〉 stacking as an apolar interaction between Y373 and W322 to be responsible for the characteristics of the tyrosyl radical. This concept of radical stabilization has been unknown to cryptochromes so far but might be highly relevant for other homologs with a tetrad of tryptophans and tyrosines as electron donors.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 29 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Sebastian Hillringhaus, Anil K. Dasanna, Gerhard Gompper, Dmitry A. Fedosov〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Invasion of erythrocytes by merozoites is an essential step for the survival and progression of malaria parasites. In order to invade red blood cells (RBCs), apicomplexan parasites have to adhere with their apex to the RBC membrane. This necessary apex-membrane contact (or alignment) is not immediately established, because the orientation of a free merozoite with respect to RBC membrane is random when an adhesion contact first occurs. Therefore, it has been suggested that after the initial adhesion, merozoites facilitate their proper alignment by inducing considerable membrane deformations, frequently observed before the invasion process. This proposition is based on a positive correlation between RBC membrane deformation and successful invasion; however, the role of RBC mechanics and its deformation in the alignment process remains elusive. Using a mechanically realistic model of a deformable RBC, we investigate numerically the importance of RBC deformability for merozoite alignment. Adhesion between the parasite and RBC membrane is modeled by an attractive potential which might be inhomogeneous, mimicking possible adhesion gradients at the surface of a parasite. Our results show that RBC membrane deformations are crucial for successful merozoite alignment, and require interaction strengths comparable to adhesion forces measured experimentally. Adhesion gradients along the parasite body further improve its alignment. Finally, an increased membrane rigidity is found to result in poor merozoite alignment, which can be a possible reason for a reduction in the invasion efficiency of RBCs in several blood diseases associated with membrane stiffening.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 21 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Orkide Ordu, Alexandra Lusser, Nynke H. Dekker〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)〈sub〉2〈/sub〉 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque build-up in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behaviour. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome, but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 21 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Heiko Heerklotz, Erwin London〈/p〉

Description: 〈p〉Publication date: Available online 22 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): J.F. Nagle〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Extraction from simulations of the area compressibility moduli of the monolayers in a bilayer is considered theoretically. A statistical mechanical derivation shows that the bilayer modulus is the sum of the two monolayer moduli, as is often supposed, but contrary to a recent study. Seemingly plausible assumptions regarding fluctuations are tested rigorously. Prospects for future research are discussed.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Jeffrey P. Bonin, Paul J. Sapienza, Emily Wilkerson, Dennis Goldfarb, Li Wang, Laura Herring, Xian Chen, Michael B. Major, Andrew L. Lee〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Thymidylate synthase (TS) catalyzes the production of the nucleotide dTMP from dUMP, making the enzyme necessary for DNA replication and consequently a target for cancer therapeutics. TSs are homodimers with active sites separated by ∼30 Å. Reports of half-the-sites activity in TSs from multiple species demonstrate the presence of allosteric communication between the active sites of this enzyme. A simple explanation for the negative allosteric regulation occurring in half-the-sites activity would be that the two substrates bind with negative cooperativity. However, previous work on 〈em〉E. coli〈/em〉 TS revealed that dUMP substrate binds without cooperativity. To gain further insight into TS allosteric function, binding cooperativity in human TS is examined here. ITC and 2D lineshape analysis of NMR titration spectra are used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperativity between the two substrate binding events. We find that human TS binds dUMP with ∼9-fold entropically-driven positive cooperativity 〈em〉ρ〈sub〉ITC〈/sub〉〈/em〉=9±1, 〈em〉ρ〈sub〉NMR〈/sub〉〈/em〉=7±1), in contrast to the apparent strong negative cooperativity reported previously. Our work further demonstrates the necessity of globally fitting isotherms collected under various conditions, as well as accurate determination of binding competent protein concentration, for calorimetric characterization of homotropic cooperative binding. Notably, an initial curvature of the isotherm is found to be indicative of positively cooperative binding. 2D lineshape analysis NMR is also found to be an informative tool for quantifying binding cooperativity, particularly in cases where bound intermediates yield unique resonances.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 21 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Rev R.L. Aure, Christopher C. Bernido, M.Victoria Carpio-Bernido, Rommel G. Bacabac〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉From observations of colloidal tracer particles in fibrin undergoing gelation, we introduce an analytical framework that allows determination of the probability density function (PDF) for a stochastic process beyond fractional Brownian motion. Using passive microrheology via videomicroscopy, mean square displacements (MSD) of tracer particles suspended in fibrin at different ageing times are obtained. The anomalous diffusion is then described by a damped white noise process with memory, with analytical results closely matching experimental plots of MSD and PDF. We further show that the white noise functional stochastic approach applied to passive microrheology reveals the existence of a gelation parameter 〈em〉μ〈/em〉 which elucidates the dynamics of constrained tracer particles embedded in a time dependent soft material. In addition, we found that microstructural heterogeneity of particle environments decreases as the ageing time increases. This study offers experimental insights on the ageing of fibrin gels while presenting a white noise functional stochastic approach that could be applied to other systems exhibiting non-Markovian diffusive behavior.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 21 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Thais A. Enoki, Gerald W. Feigenson〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of GUVs and a supported lipid bilayer (SLB), triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all new exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of GUVs composed of DSPC/DOPC/chol = 0.39/0.39/0.22 and a SLB of DOPC/chol = 0.8/0.2 we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe C-Laurdan, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (〉 70%) the dye partition indicates induced-disordered and -ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric Ld and Lo domains.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Anindita Das, Satyaki Chatterjee, H. Raghuraman〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Voltage-gated potassium (K〈sub〉v〈/sub〉) channels play a fundamental role in neuronal and cardiac excitability and are potential therapeutic targets. They assemble as tetramers with a centrally located pore-domain surrounded by a voltage-sensing domain (VSD), which is critical for sensing transmembrane potential and subsequent gating. Although the sensor is supposed to be in ‘Up’ conformation in both n-octylglucoside (OG) micelles and phospholipid membranes in the absence of membrane potential, toxins that bind VSD and modulate the gating behaviour of K〈sub〉v〈/sub〉 channels exhibit dramatic affinity differences in these membrane-mimetic systems. In this study, we have monitored the structural dynamics of the S3b-S4 loop of the paddle motif in activated conformation of KvAP-VSD by site-directed fluorescence approaches, using the environment-sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD). Emission maximum of NBD-labeled loop region of KvAP-VSD (residues 110 to 117) suggests a significant change in the polarity of local environment in POPC/POPG membranes compared to OG micelles. This indicates that S3b-S4 loop residues might be partitioning to membrane interface, which is supported by an overall increased mean fluorescence lifetimes and significantly reduced water accessibility in membranes. Further, the magnitude of Red Edge Excitation Shift (REES) support the presence of restricted/bound water molecules in the loop region of the VSD in micelles and membranes. Quantitative analysis of REES data using Gaussian probability distribution function clearly indicates that the sensor loop has lesser number of discrete equilibrium conformational states when reconstituted in membranes. Interestingly, this reduced molecular heterogeneity is consistent with the site-specific NBD polarization results, which suggest that the membrane environment offers a relaxed/dynamic organization for most of the S3b-S4 loop residues of the sensor. Overall, our results are relevant for understanding toxin-VSD interaction and gating mechanisms of K〈sub〉v〈/sub〉 channels in membranes.〈/p〉〈/div〉