Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry
Biophys J. 2023 Nov 17:S0006-3495(23)00719-1. doi: 10.1016/j.bpj.2023.11.019. Online ahead of print.ABSTRACTMeasuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed non-fluorescent membrane scaffold protein (MSP) to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). ...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: Jazlyn A Selvasingh Eli F McDonald Preston D Neufer Jacob R McKinney Jens Meiler Kaitlyn V Ledwitch Source Type: research

Conformational transitions of the HIV-1 Gag polyprotein upon multimerization and gRNA binding
Biophys J. 2023 Nov 17:S0006-3495(23)00717-8. doi: 10.1016/j.bpj.2023.11.017. Online ahead of print.ABSTRACTDuring the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-gRNA interactions play a crucial role in the multimerization process, which is yet to be fully understood. We have performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model (hENM) a...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: Puja Banerjee Gregory A Voth Source Type: research

Disruption of the FMN-A524 interaction cascade and Glu513 induced collapse of the hydrophobic barrier promotes light induced J α-helix unfolding in AsLOV2
This study indicates that the displacement of N492 out of the FMN binding pocket, not necessarily requiring Q513, is essential for the initiation of the Jα-helix unfolding. Rather, the structural reorientation of Q513 activates the protein to cross the hydrophobic barrier and enter the post initiation phase. Similarly, the structural deviations in N482, rather than its integral role in unfolding, could enhance the unfolding rates. Further, the MSM studies on the wild type and the Q513 mutant, provide the spatio-temporal roadmap that layout the possible pathways of structural transition between the dark and the light state...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: Syeda Amna Arshi Manisha Chauhan Amit Sharma Source Type: research

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI
Biophys J. 2023 Nov 17:S0006-3495(23)00718-X. doi: 10.1016/j.bpj.2023.11.018. Online ahead of print.ABSTRACTRho-specific guanine dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code", where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Ra...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: Krishnendu Sinha Amit Kumawat Hyunbum Jang Ruth Nussinov Suman Chakrabarty Source Type: research

Quantifying single cell diacylglycerol signaling kinetics after uncaging
Biophys J. 2023 Nov 16:S0006-3495(23)00713-0. doi: 10.1016/j.bpj.2023.11.013. Online ahead of print.ABSTRACTStudying the role of molecularly distinct lipid species in cell signaling remains challenging due to a scarcity of methods for performing quantitative lipid biochemistry in living cells. We have recently used lipid uncaging to quantify lipid-protein affinities and rates of lipid transbilayer movement and turnover in the diacylglycerol signaling pathway. This approach is based on acquiring live-cell dose-response curves requiring light dose titrations and experimental determination of uncaging photoreaction efficiency...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: David T Gonzales Milena Schuhmacher H Mathilda Lennartz Juan M Iglesias-Artola Sascha M Kuhn Pavel Barahtjan Christoph Zechner Andr é Nadler Source Type: research

Lamellipodia Dynamics and Microrheology in Endothelial Cell Paracellular Gap Closure
In this study, we extend our earlier work examining the biophysical properties of EC gap formation and now address the role of lamellipodia, thin sheet-like membrane projections from the leading edge, in modulating EC spatial-specific contractile properties and gap closure. Micropillars, fabricated by soft lithography, were utilized to form reproducible paracellular gaps in human lung EC. Using time lapse imaging via optical microscopy, rates of EC gap closure and motility were measured with and without EC stimulation with the barrier-enhancing sphingolipid, sphingosine-1-phosphate (S1P). Peripheral ruffle formation was ub...
Source: Biophysical Journal - November 18, 2023 Category: Physics Authors: Fernando Teran Arce Scott Younger Amir A Gaber Joseph B Mascarenhas Marisela Rodriguez Steven M Dudek Joe G N Garcia Source Type: research

The influence of coiled-coil motif of serine recombinase toward the directionality regulation
Biophys J. 2023 Nov 16:S0006-3495(23)00709-9. doi: 10.1016/j.bpj.2023.11.009. Online ahead of print.ABSTRACTSerine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor (RDF). We utilized Tethered Particle Motion (TPM) experiments to investigate the reaction behaviours of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carri...
Source: Biophysical Journal - November 17, 2023 Category: Physics Authors: Yei-Wei Chen Bo-Yu Su Gregory D Van Duyne Paul Fogg Hsiu-Fang Fan Source Type: research

Differentiating the roles of proteins and polysomes in nucleoid size homeostasis in Escherichia coli
Biophys J. 2023 Nov 16:S0006-3495(23)00710-5. doi: 10.1016/j.bpj.2023.11.010. Online ahead of print.ABSTRACTA defining feature of the bacterial cytosolic interior is a distinct membrane-less organelle, the nucleoid, that contains the chromosomal DNA. Although increasing experimental evidence indicates that macromolecular crowding is the dominant mechanism for nucleoid formation, it has remained unclear which crowders control nucleoid volume. It is commonly assumed that polyribosomes play a dominant role, yet the volume fraction of soluble proteins in the cytosol is comparable to that of polyribosomes. Here, we develop a fr...
Source: Biophysical Journal - November 17, 2023 Category: Physics Authors: Mu-Hung Chang Maxim O Lavrentovich Jaan M ännik Source Type: research

The influence of coiled-coil motif of serine recombinase toward the directionality regulation
Biophys J. 2023 Nov 16:S0006-3495(23)00709-9. doi: 10.1016/j.bpj.2023.11.009. Online ahead of print.ABSTRACTSerine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor (RDF). We utilized Tethered Particle Motion (TPM) experiments to investigate the reaction behaviours of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carri...
Source: Biophysical Journal - November 17, 2023 Category: Physics Authors: Yei-Wei Chen Bo-Yu Su Gregory D Van Duyne Paul Fogg Hsiu-Fang Fan Source Type: research

Differentiating the roles of proteins and polysomes in nucleoid size homeostasis in Escherichia coli
Biophys J. 2023 Nov 16:S0006-3495(23)00710-5. doi: 10.1016/j.bpj.2023.11.010. Online ahead of print.ABSTRACTA defining feature of the bacterial cytosolic interior is a distinct membrane-less organelle, the nucleoid, that contains the chromosomal DNA. Although increasing experimental evidence indicates that macromolecular crowding is the dominant mechanism for nucleoid formation, it has remained unclear which crowders control nucleoid volume. It is commonly assumed that polyribosomes play a dominant role, yet the volume fraction of soluble proteins in the cytosol is comparable to that of polyribosomes. Here, we develop a fr...
Source: Biophysical Journal - November 17, 2023 Category: Physics Authors: Mu-Hung Chang Maxim O Lavrentovich Jaan M ännik Source Type: research

De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features
Biophys J. 2023 Nov 15:S0006-3495(23)00708-7. doi: 10.1016/j.bpj.2023.11.008. Online ahead of print.ABSTRACTDNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/non-canonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/non-canonical structures into plasmids often requires several rounds of cloning on pre...
Source: Biophysical Journal - November 16, 2023 Category: Physics Authors: Daniel Ram írez Montero Zhaowei Liu Nynke H Dekker Source Type: research

De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features
Biophys J. 2023 Nov 15:S0006-3495(23)00708-7. doi: 10.1016/j.bpj.2023.11.008. Online ahead of print.ABSTRACTDNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/non-canonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/non-canonical structures into plasmids often requires several rounds of cloning on pre...
Source: Biophysical Journal - November 16, 2023 Category: Physics Authors: Daniel Ram írez Montero Zhaowei Liu Nynke H Dekker Source Type: research

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity
Biophys J. 2023 Nov 9:S0006-3495(23)00691-4. doi: 10.1016/j.bpj.2023.11.006. Online ahead of print.ABSTRACTMost antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMPs-induced cell membrane damage in Escherichia coli and AMPs-induced cell death at the single-cell level. Magainin 2, lactofer...
Source: Biophysical Journal - November 11, 2023 Category: Physics Authors: Md Zahidul Islam Farzana Hossain Md Hazrat Ali Masahito Yamazaki Source Type: research

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity
Biophys J. 2023 Nov 9:S0006-3495(23)00691-4. doi: 10.1016/j.bpj.2023.11.006. Online ahead of print.ABSTRACTMost antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMPs-induced cell membrane damage in Escherichia coli and AMPs-induced cell death at the single-cell level. Magainin 2, lactofer...
Source: Biophysical Journal - November 11, 2023 Category: Physics Authors: Md Zahidul Islam Farzana Hossain Md Hazrat Ali Masahito Yamazaki Source Type: research

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity
Biophys J. 2023 Nov 9:S0006-3495(23)00691-4. doi: 10.1016/j.bpj.2023.11.006. Online ahead of print.ABSTRACTMost antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMPs-induced cell membrane damage in Escherichia coli and AMPs-induced cell death at the single-cell level. Magainin 2, lactofer...
Source: Biophysical Journal - November 11, 2023 Category: Physics Authors: Md Zahidul Islam Farzana Hossain Md Hazrat Ali Masahito Yamazaki Source Type: research