Biophysical Investigations Research Articles

Calmodulin extracts the ras family protein rala from lipid bilayers by engagement with two membrane-targeting motifs.

RalA is a membrane-attached small GTPase being crucial for tumorigenesis and uncontrolled growth in Ras-driven cancers. Its function as molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. It has been shown before that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. In this work, using a range of structural and biophysical methods (nuclear magnetic resonance, isothermal titration calorimetry and surface plasmon resonance), we have solved the structure of calmodulin in complex with the lipidated C-terminal tail of RalA and show that complex formation leads to RalA being removed from the lipid bilayer. We found that CaM interacts exclusively with the C-terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Furthermore, we show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B aiding to find new ways of targeting GTPases in cancer. (1) ((1) DOI: https://doi.org/10.1073/pnas.2104219118).

Unraveling the phase separation of EWS low complexity domain using molecular dynamics simulations.

EWS, an RNA-binding protein, is responsible for gene expression, cell signaling, RNA processing, and transport. It is a member of the FET (FUS, EWS, TAF15) family, whose derailed phase separation is known to cause many neurodegenerative diseases. Through achromosomal translocation, the low complexity domain (LCD) of EWS may be fused to the DNA binding domain of the transcription factor FLI1. The resultant EWS-FLI1 fusion is a potent oncogenic protein responsible for the development and growth of Ewing sarcoma and aggressive bone cancer, often affecting adolescents and young adults. Though studies have shown that the LCD is capable of liquid-liquid phase separation (LLPS), mechanistic details are still lacking. In this study, using molecular dynamics simulations, we aim to understand at atomic resolution the mechanism underpinning phase separation of the EWS-LCD. We have performed single-chain all-atom simulations and multi-chain coarse-grain simulations to investigate the intra and interchain contacts responsible for LLPS of EWS-LCD. Our study reveals that Tyrosine and Glutamine residues form contacts with the greatest frequency in both single and multi-chain interactions. We observed that residues near the C-terminus are more likely to form contacts than N-terminal residues. Our results are consistent with experimental results derived from biophysical investigations. This study is a step towards understanding the self-association of the EWS-LCD at the atomic level, and it will provide insight into the specific interactions that drive EWS phase separation.

A novel high throughput combined voltage-clamp/current-clamp analysis of single primary neurons.

The patch-clamp technique is the gold-standard methodology for biophysical investigations of channels and receptors studied by voltage-clamp, and for analysis of excitability of cells, such as neurons studied via current-clamp. However, the throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for assessments, such as drug screening, has been primarily used to study channels and receptors expressed in heterologous expression systems. In this study, we introduce a novel approach to automated high throughput patch-clamping that substantially enhances high throughput analysis of excitable cells at the channel and cellular level. As a proof-of-concept, we apply this approach to investigate the detailed biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables high throughput, unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of primary neurons. Furthermore, our approach eliminates the need for culturing of cells on coverslips and provides the ability to perform both voltage- and current-clamp recordings on the same neuron. This approach can be used for many applications, including both physiological and pharmacological analyses of primary DRG neurons.

A single molecule TIRF-based platform to study DNA supercoiling effect on DNA processing enzymes.

DNA topology plays an important role in regulating DNA metabolic pathways. DNA supercoiling changes the DNA properties and proteins may differentially interact with DNA under various supercoiling states. A biophysical investigation of how proteins differentiate various topological states could further our understanding of protein-DNA interactions. We developed new single molecule total internal reflection fluorescence (smTIRF) -based platform to investigate how DNA topology affects protein-DNA interactions. We used this platform to study MutS, which detects a mismatched base pair to initiate mismatch repair, and CRISPR-Cas9. We generated plasmids that are site-specifically labeled with one or two fluorophores and a biotin. We prepared them in three different topological states: “Linear”, “relaxed circular”, and “negatively supercoiled”. For MutS study, we designed a Cy5 labeled plasmid with an adjacent mismatch. We observed FRET between Cy3-MutS and Cy5 when MutS bound the mismatch. In the presence of ADP, MutS bound the negative supercoiled DNA with a longer residence time compared to the relaxed circular DNA, suggesting that negative supercoiling helps mismatch recognition. For CRISPR-Cas9, we created a dual labeled plasmid to observe R-loop formation induced by dCas9. R-loop formation rate was similar for all three DNA topological states when the guide RNA sequence matched the target DNA sequence, while the rate was highest for the negatively supercoiled DNA and decreased progressively for the relaxed circular DNA and the linear DNA when we introduced one mismatch in either the PAM (protospacer adjacent motif)-proximal position or the PAM-distal position. Our observation confirms that DNA negative supercoiling assists DNA unwinding by Cas9, and this can exacerbate off-target effects in the cell. Collectively, our smTIRF-based platform allows high throughput investigation of supercoiling effects on DNA protein interactions as demonstrated using two different biological systems.

Biophysical investigations of antimicrobial peptide mimics for mechanistic studies.

With a relentless rise in the growth of antimicrobial resistance, much attention is being focused on Antimicrobial peptides (AMP) and the development of AMP mimicking agents to combat this nefarious issue. However lesser reports come up with adequate reasoning behind the driving force of the membrane perturbing nature. Here, we shall be discussing the two branches of AMP mimics, peptoids and small molecules. Further, we explore the mode of action behind the antimicrobial properties displayed by the library of peptoids and small molecule mimics. To address this big challenge of understanding how the membrane active compound behaves, each of the compounds were introduced to the liposomes, mimicking the bacterial model membranes, and their interactions was recorded using solid state nuclear magnetic resonance spectroscopy. We observe that each of the mimics interact with the liposome as the global shape of the vesicles gets deformed. Also we observed a definite interaction with the fatty acyl chain as changes in order parameters were observed with respect to that of pure lipids. To develop a thorough understanding of the pore forming abilities of these compounds, a fluorescent dye release studies was executed using both bacterial and mammalian model membranes. The calcein dye release test revealed that the small molecule mimics undergo a cooperative effect. For the peptoids investigated, the extent of release could be correlated with their antimicrobial activity as well as the toxicity data.

Getting to the heart of it - Small GTPase activity probed at an atomic level in cells.

Ras homology protein (RhoA), a small G protein (SmG), plays a crucial pathophysiological role and is implicated in cardiovascular diseases such as diabetes, heart failure, hypertension, and stroke. RhoA regulates cytoskeleton remodeling through actin fibrillation and aids in overall cell survival. Like all SmGs that function as regulatory switches, the amount of RhoA in the heart is delicately controlled, with large changes resulting in faulty signaling that can lead to cardiac disease. ∼2-fold overexpression protects myocytes from ischemic injury and decreases fibrosis during the stress response, while a large excess (∼20-fold) is associated with cardiac hypertrophy, fibrosis, and cardiomyopathy. A myriad of studies have been conducted on the structure-function relationship of SmGs, with most structural biology and biophysical investigations employing a reductionist in vitro strategy in which the native physiological context is ignored. In order to assess how proteins work in vivo, there is a growing emphasis to investigate systems in a native physiological environment. In this presentation, I will report on my investigation of conformational dynamics and enzymatic activity of the RhoA protein in a crowded environment. I will discuss RhoA behavior using 2D 15N-1H NMR, 19F NMR and fluorescence spectroscopy and will present my findings on both the enzyme activity and conformations of GDP- and GTP- bound RhoA under the influence of macromolecular crowders, small molecule metabolite crowding, and the role of cardio-specific metabolites. In particular, I will use 19F nuclei as robust cellular NMR tracers and reporters on live in-cell using19F labeled RhoA protein

Mechanosensitive Channel-Based Optical Membrane Tension Reporter.

Plasma membrane tension functions as a global physical organizer of cellular activities. Technical limitations of current membrane tension measurement techniques have hampered in-depth investigation of cellular membrane biophysics and the role of plasma membrane tension in regulating cellular processes. Here, we develop an optical membrane tension reporter by repurposing an E. coli mechanosensitive channel via insertion of circularly permuted GFP (cpGFP), which undergoes a large conformational rearrangement associated with channel activation and thus fluorescence intensity changes under increased membrane tension.

Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors.

Among its many molecular targets, the ubiquitous calcium sensor protein calmodulin (CaM) recognizes and regulates the activity of ryanodine receptors type 1 (RyR1) and 2 (RyR2), mainly expressed in skeletal and cardiac muscle, respectively. Such regulation is essential to achieve controlled contraction of muscle cells. To unravel the molecular mechanisms underlying the target recognition process, we conducted a comprehensive biophysical investigation of the interaction between two calmodulin variants associated with congenital arrhythmia, namely N97I and Q135P, and a highly conserved calmodulin-binding region in RyR1 and RyR2. The structural, thermodynamic, and kinetic properties of protein-peptide interactions were assessed together with an in-depth structural and topological investigation based on molecular dynamics simulations. This integrated approach allowed us to identify amino acids that are crucial in mediating allosteric processes, which enable high selectivity in molecular target recognition. Our results suggest that the ability of calmodulin to discriminate between RyR1 an RyR2 targets depends on kinetic discrimination and robust allosteric communication between Ca2+-binding sites (EF1-EF3 and EF3-EF4 pairs), which is perturbed in both N97I and Q135P arrhythmia-associated variants.

N,N′,N′′-Trisubstituted guanidine derivatives as DNA-intercalators: synthesis, crystal structures and biophysical investigations

Biophysical and metadynamics simulation studies indicated partial intercalation of one of the phenyl rings of N,N′,N′′-trisubstituted guanidine derivatives between the base pairs of DNA.

Abstract 2726: Biophysical and In-Silico Investigation of Polyphenols Targeting Bifunctional DAH7PS from Bacillus subtilis

Abstract 2726: Biophysical and In-Silico Investigation of Polyphenols Targeting Bifunctional DAH7PS from Bacillus subtilis

Prevalence of Chronic Kidney Disease Among Diabetes and Hypertensive Patients in a Teaching Hospital in Ekiti State, Southwest Nigeria

Introduction: Chronic kidney disease (CKD) is a growing public health problem associated with enormous economic burdens, reduced quality of life, and untimely deaths, predominantly in developing countries. Aims: The study determines the prevalence and risk factors for CKD among diabetes and hypertensive patients in a teaching hospital in Ekiti State. Methods: Descriptive and cross-sectional research designs were employed using a quantitative strategy. Two hundred (200) randomly selected participants participated in the study. Socio-demographic data, awareness, and risk factors for CKD were determined using a standardized questionnaire, while CKD prevalence was investigated with biophysical measurements and laboratory investigations. Descriptive analyses were used to answer the research questions, while inferential statistics were used to test hypotheses at a significant level of p < 0.05. Results: Findings revealed that 50% and 57.1% of the diabetics and hypertensives were above 60 years, 36.7% of the people with diabetes had comorbidity, while only 2% and 3.1% of diabetics and hypertensives participants had a family history of CKD. The study revealed that the respondents' level of awareness of CKD was inadequate. Major risk factors of CKD identified among the respondents were already diagnosed with diabetes and hypertension, age above 60 years (50% and 57.1%), herbal concoction (77.7% and 73.5%), and NSAID (74.5% and 78.6%). The prevalence of CKD for people with diabetes was 39.8%, while 57.1% for hypertensives. There was a significant relationship between respondents’ level of education and awareness of CKD (X2 =44.20, p=<0.001). The prevalence of CKD among the studied population was high. Conclusion: Efforts should be intensified by nurses and all other stakeholders on awareness and prevention programs for CKD. Furthermore, the promotion of patients’ satisfaction with the quality of healthcare services should be the goal to promote positive health outcomes.

Biophysical investigation of the interaction between NSAID ibuprofen and cationic biodegradable Cm-E2O2-Cm gemini surfactants

The surfactant-based drug delivery phenomenon has recently received tremendous interest from researchers and pharmaceutical industries. Moreover, a significant shift has been recorded from conventional surfactants to gemini surfactants owing to the advanced chemical and environmental properties of the latter. They are also found to be easily tunable to suit the research and industry requirements. In this work, we have explored the binding interactivity of the non-steroidal anti-inflammatory drug Ibuprofen (IBF) with finely tuned di-ester-based cationic Cm-E2O2-Cm gemini surfactants (where m = alkyl chain length, viz. 12, 14, and 16 and E2O2 is di-ester based spacer) below and above CMC levels. Modification in the surface and interfacial properties of the geminis occurred showing the presence of an interaction between the participants of the studied systems. Fluorescence spectroscopy revealed that the Stern-Volmer constant (Ksv) and binding constant (Kb) follow the same order, which is C12-E2O2-C12 < C16-E2O2-C16 < C14-E2O2-C14 above the CMC but the order changes in the pre-micellar region as C12-E2O2-C12 < C14-E2O2-C14 < C16-E2O2-C16 for Ksv while Kb follows an inverse order. The obtained thermodynamic parameters suggested spontaneous interaction between the examined entities. UV–vis spectroscopic studies (both below and above CMC levels) confirmed the obtained fluorescence results. Additionally, the microenvironment of IBF is changed in the presence of surfactants, suggesting IBF-Cm-E2O2-Cm complexation. FT-IR spectroscopic study also suggests the drug-surfactant complexation. HOMO-LUMO energy gap calculated by the DFT method deduced the feasibility of the interaction. The MD simulation calculations were carried out to find different kind of energies involved in binding process.

Structure and Dynamics of Human Chemokine CCL16-Implications for Biological Activity.

Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.

Protein delivery to living cells by thermal stimulation for biophysical investigation

Studying biomolecules in their native environment represents the ideal sample condition for structural biology investigations. Here we present a novel protocol which allows to delivery proteins into eukaryotic cells through a mild thermal stimulation. The data presented herein show the efficacy of this approach for delivering proteins in the intracellular environment of mammalian cells reaching a concentration range suitable for successfully applying biophysical methods, such as double electron electron resonance (DEER) measurements for characterising protein conformations.

Molecular rec§ognition of telomere DNA sequence by 2, 6 anthraquinone derivatives leads to thermal stabilization and induces apoptosis in cancer cells

According to current research, anti-cancer anthraquinones impact telomere disruption and may interact with G-quadruplex DNA that triggers signaling to apoptosis. The present study represents the biophysical investigation of oxidative stress, late apoptosis, and induced senescence among cancer cells after binding laboratory synthesized piperidine-based anthraquinone derivatives, 2, 6- Bis [(3-piperidino)acetamido)]anthracene-9,10-dione (N1P) and 2, 6-Bis [piperidino)propionamido]anthracene-9,10-dione (N2P), with G-quadruplex DNA. We employed biophysical approaches to explore the interaction of synthetic anthraquinone derivatives with quadruplex DNA sequences to influence biological activities in the presence of K+ and Na+ cations. The binding affinity for N2P and N1P are Kb = 5.8 × 106 M−1 and Kb = 1.0 × 106 M−1, respectively, leading to hypo-/hyper-chromism with 5–7 nm red shift and significant fluorescence quenching and changes in ellipticity resulting in external binding of both the ligands to G-quadruplex DNA. Ligand binding induced enhancement of thermostability of G4 DNA is greater in Na+ environment (ΔTm = 34 °C) as compared to that in K+ environment (ΔTm = 21 °C), thereby restricting telomerase binding access to telomeres. Microscopic images of treated cells indicated cellular shape, nuclear condensation, and fragmentation alterations. The findings pave the path for therapeutic research, given the great potential of modifying anthraquinone substituent groups towards improved efficacy, ROS generation, and G-quadruplex DNA selectivity.

Integrate thermostabilized fusion protein apocytochrome b 562 RIL and N-glycosylation mutations: A novel approach to heterologous expression of human UDP-glucuronosyltransferase (UGT) 2B7.

Human UDP-glucuronosyltransferase (UGT) 2B7 is a crucial phase II metabolic enzyme that transfers glucuronic acid from UDP-glucuronic acid (UDPGA) to endobiotic and xenobiotic substrates. Biophysical and biochemical investigations of UGT2B7 are hampered by the challenge of the integral membrane protein purification. This study focused on the expression and purification of recombinant UGT2B7 by optimizing the insertion sites for the thermostabilized fusion protein apocytochrome b 562RIL (BRIL) and various mutations to improve the protein yields and homogeneity. Preparation of the recombinant proteins with high purity accelerated the measurement of pharmacokinetic parameters of UGT2B7. The dissociation constants (K D) of two classical substrates (zidovudine and androsterone) and two inhibitors (schisanhenol and hesperetin) of UGT2B7 were determined using the surface plasmon resonance spectroscopy (SPR) for the first time. Using negative-staining transmission electron microscopy (TEM), UGT2B7 protein particles were characterized, which could be useful for further exploring its three-dimensional structure. The methods described in this study could be broadly applied to other UGTs and are expected to provide the basis for the exploration of metabolic enzyme kinetics, the mechanisms of drug metabolisms and drug interactions, changes in pharmacokinetics, and pharmacodynamics studies in vitro.

Excited-State Intramolecular Hydrogen Transfer of Compact Molecules Controls Amyloid Aggregation Profiles.

Developing chemical methodologies to directly modify harmful biomolecules affords the mitigation of their toxicity by persistent changes in their properties and structures. Here we report compact photosensitizers composed of the anthraquinone (AQ) backbone that undergo excited-state intramolecular hydrogen transfer, effectively oxidize amyloidogenic peptides, and, subsequently, alter their aggregation pathways. Density functional theory calculations showed that the appropriate position of the hydroxyl groups in the AQ backbone and the consequent intramolecular hydrogen transfer can facilitate the energy transfer to triplet oxygen. Biochemical and biophysical investigations confirmed that these photoactive chemical reagents can oxidatively vary both metal-free amyloid-β (Aβ) and metal-bound Aβ, thereby redirecting their on-pathway aggregation into off-pathway as well as disassembling their preformed aggregates. Moreover, the in vivo histochemical analysis of Aβ species produced upon photoactivation of the most promising candidate demonstrated that they do not aggregate into oligomeric or fibrillar aggregates in the brain. Overall, our combined computational and experimental studies validate a light-based approach for designing small molecules, with minimum structural complexity, as chemical reagents targeting and controlling amyloidogenic peptides associated with neurodegenerative disorders.

Bioengineered lipophilic Ru(III) complexes as potential anticancer agents.

Lipid-conjugated Ru(III) complexes - designed to obtain lipophilic analogues of the low molecular weight derivative AziRu, which is a NAMI-A-like anticancer agent - have been synthesized and fully characterized. A detailed biophysical investigation, including multiple, integrated techniques, allowed determining their molecular and self-assembling properties in aqueous solutions mimicking the extracellular environment, showing that our design produced a protective effect from hydrolysis of the Ru(III) complexes. In vitro biological experiments, carried out in comparison with AziRu, demonstrated that, among the novel lipophilic Ru(III) complexes synthesized, the compounds derivatized with palmitic and stearic acid, that we named PalmiPyRu and StePyRu respectively, showed attractive features and a promising antiproliferative activity, selective on specific breast cancer phenotypes. To get a deeper insight into their interactions with potential biomacromolecular targets, their ability to bind both bovine serum albumin (BSA), an abundant serum carrier protein, and some DNA model systems, including duplex and G-quadruplex structures, has been investigated by spectroscopic techniques. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis of the ruthenium amount incorporated in human MCF-7 and MDA-MB-231 breast cancer cells, after incubation in parallel experiments with PalmiPyRu and AziRu, showed a markedly higher cell uptake of the lipophilic Ru(III) complex with respect to AziRu. These data confirmed that the proper lipidic tail decorating the metal complex not only favoured the formation of aggregates in the extracellular media but also improved their cell membrane penetration, thus leading to higher antiproliferative activity selective on breast cancer cells.

Yoda1’s energetic footprint on Piezo1 channels and its modulation by voltage and temperature

Piezo1 channels are essential mechanically activated ion channels in vertebrates. Their selective activation by the synthetic chemical activator Yoda1 opened new avenues to probe their gating mechanisms and develop novel pharmaceuticals. Yet, the nature and extent of Piezo1 functions modulated by this small molecule remain unclear. Here we close this gap by conducting a comprehensive biophysical investigation of the effects of Yoda1 on mouse Piezo1 in mammalian cells. Using calcium imaging, we first show that cysteine bridges known to inhibit mechanically evoked Piezo1 currents also inhibit activation by Yoda1, suggesting Yoda1 acts by energetically modulating mechanosensory domains. The presence of Yoda1 alters single-channel dwell times and macroscopic kinetics consistent with a dual and reciprocal energetic modulation of open and shut states. Critically, we further discovered that the electrophysiological effects of Yoda1 depend on membrane potential and temperature, two other Piezo1 modulators. This work illuminates a complex interplay between physical and chemical modulators of Piezo1 channels.

Evaluation of Hepatic, Renal and Cerebral Function Test in Preeclampsia-Eclampsia Syndrome and its Correlation to Maternal and Fetal Outcome

Introduction: Hypertensive disorders complicates 5-10% of pregnancies all over the world and its incidence in India was found to be 10.08% as per data of National Eclampsia Registry (NEP).&#x0D; Objectives: The objectives of this study were to determine the incidence of abnormal liver, renal and cerebral function test among patients with preeclampsia and eclampsia and their correlation to maternal and fetal outcome.&#x0D; Methodology: The study was a prospective cohort study comparing the maternal and fetal outcomes of preeclampsia and eclampsia patients with abnormal parameters (cases; n=50) and pre-eclampsia and eclampsia patients with normal parameters (controls; n=50). Both case and control were examined clinically apart from biophysical and biochemical investigation.&#x0D; Results: Deranged LFT was present in approximately 56% -70% of cases. Serum albumin was decreased in 80% of cases. Prothrombin time (PT) was raised in 48% of cases. Abnormal GFR, urea, creatinine and uric acid level were present in 44%, 10%, 10% and 64% of cases. Among all the pregnancy outcomes in preeclampsia and eclampsia cases with abnormal LFT, preterm labour, PPH, IUFD, meconium stained liquor and neonatal death had significant “p” value &lt;.05. There were 16% preterm labours, 60% IUGR, 36% ARF, 18% Neonatal death, in cases with abnormal RFT. It was found from the study that CVA, Cerebral hemorrhage, fetal distress, and still birth were present in 16%, 24%, 36% and 8% of cases with abnormal cerebral function.&#x0D; Conclusion: Deranged liver function test was associated with increased incidence of postpartum hemorrhage (P value=0.0001) and postpartum eclampsia (P value &lt;0.0001).