Biology subjects

Freed, J. H.

Publications and source records attributed to Freed, J. H..

10 recordsLinked to original sources

bioRxiv2025

Localized Reconstruction of Multimodal Distance Distribution from DEER Data of Biopolymers

Pulsed Dipolar ESR Spectroscopy (PDS) is a uniquely powerful technique to characterize the structural property of intrinsically disordered proteins (IDPs) and polymers and the conformational evolution of IDPs and polymers, e.g. during assembly, by offering the probability distribution of segment end-to-end distances. However, it is challenging to determine distance distribution P(r) of IDPs by PDS because of the uncertain and broad shape information that is intrinsic to the distance distribution of IDPs. We demonstrate here that the Srivastava-Freed Singular Value Decomposition (SF-SVD) point-wise mathematical inversion method along with wavelet denoising (WavPDS) can aid in obtaining reliable shapes for the distance distribution, P(r), for IDPs. We show that broad regions of P(r) as well as mixed narrow and broad features within the captured distance distribution range can be effectively resolved and differentiated without a priori knowledge. The advantage of SF-SVD and WavPDS is that the methods are transparent, requiring no adjustable parameters, the processing of the magnitude for the probability distribution is performed separately for each distance increment, and the outcome of the analysis is independent of the users judgement. We demonstrate the performance and present the application of WavPDS and SF-SVD on model ruler molecules, model polyethylene glycol polymers with end-to-end spin labeling, and IDPs with pairwise labeling spanning different segments of the protein tau to generate the transparent solutions to the P(r)s including their uncertainties and error analysis.

biophysics↗

bioRxiv2024

Flavoproteins as native and genetically encoded spin probes for in cell ESR spectroscopy

Flavin cofactors are attractive Electron Spin Resonance (ESR) probes for proteins because cellular reductants and light can generate their semiquinone states. We have used ESR spectroscopy to study the bacterial transmembrane aerotaxis receptor (Aer) in its native Escherichia coli membrane environment. Optimization of the spectroscopic (electronic relaxation times) and cell growth (isotopic labeling) conditions allowed for measurements of Aer with its partners - the histidine kinase (CheA) and the coupling protein (CheW) - in native signaling arrays. Continuous-wave ESR measurements at room temperature showed a rigid Aer flavin immobilized in the cofactor pocket and Q-band electron nuclear double resonance (ENDOR) measurements identified a predominant anionic semiquinone radical state in cell. Q-band four-pulse double electron-electron resonance (4P-DEER) measurements indicated a 4.1 nm distance between the two flavins of an Aer homodimer, consistent with previous in vitro measurements, but also revealed additional separations in cell indicative of chemoreceptor arrays, not previously observed for Aer. For general application, we further developed a genetically encoded Light-Oxygen and Voltage (LOV) domain for incorporation into target proteins as an ESR probe of structural properties in cell. This approach provides a framework to elucidate protein oligomeric states and conformations that are difficult to reproduce in vitro.

biochemistry↗

bioRxiv2023

The crystal structure of bacteriophage lambda RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on E. coli {lambda} phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the {lambda} bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here we present the 2.05-[A] crystal structure of the {lambda} RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

microbiology↗

bioRxiv2023

Signal Transduction and Kinase Control in Nanodisc Reconstituted E. coli Aerotaxis Receptor

WITHDRAWAL STATEMENTThe authors have withdrawn this manuscript because its being revised. The authors wish to withdraw this manuscript for two reasons. Firstly, the relevance and validity of some of the mass spectrometry data in the manuscript are being questioned by the authors. Secondly, some of the authors had not adequately reviewed the manuscript before it was uploaded to bioRxiv. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

biophysics↗

bioRxiv2022

Structure and dynamics of human perilipin 3 membrane association

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

biochemistry↗

bioRxiv2021

Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV ("SARS-1"), SARS-CoV-2 ("SARS-2") and MERS-CoV ("MERS"). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2 cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion. In this study, we want to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.

molecular biology↗

bioRxiv2021

Highly basic clusters in the HSV-1 nuclear egress complex drive membrane budding by inducing lipid ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in-vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an "off" switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

microbiology↗

bioRxiv2021

SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2 cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue difference, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.

molecular biology↗

bioRxiv2020

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

biochemistry↗

bioRxiv2019

Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging zoonotic infectious disease. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans with 2,468 confirmed cases, causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. A critical step in the life cycle of MERS-CoV is the fusion with the host cell with its spike (S) protein as main determinant of viral entry. Proteolytic cleavage of S exposes its fusion peptide (FP), which initiates membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium (Ca2+) plays an important role for fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FP share a high sequence homology and here, we investigated whether Ca2+ is required for MERS-CoV fusion by substituting E and D residues in the MERS-CoV FP with neutrally charged alanines. Upon verifying mutant cell surface expression and proteolytic cleavage, we tested the mutants ability to mediate infection of pseudo-particles (PPs) on host cells without and with Ca2+. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV WT PPs infection by approximately two-fold and that E891 is a crucial residue for Ca2+ interaction. Electron spin resonance revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry titration showed that MERS-CoV FP binds one Ca2+, as opposed to SARS-CoV FP which binds two. Our data suggests that there are significant differences in FP-Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity and that the number of Ca2+ ions interacting with the FP has implications on the fusion dynamics of the virus.

microbiology↗