Protein Chemistry Research Articles

FeII Metallohelices Stabilize DNA G-Quadruplexes and Downregulate the Expression of G-Quadruplex-Regulated Oncogenes.

DNA G-quadruplexes (G4s) have been identified within the promoter regions of many proto-oncogenes. Thus, G4s represent attractive targets for cancer therapy, and the design and development of new drugs as G4 binders is a very active field of medicinal chemistry. Here, molecular biophysics and biology methods were employed to investigate the interaction of chiral metallohelices with a series of four DNA G4s (hTelo, c-myc, c-kit1, c-kit2) that are formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC and c-KIT proto-oncogenes. We show that the investigated water-compatible, optically pure metallohelices, which are made by self-assembly of simple nonpeptidic organic components around FeII ions and exhibit bioactivity emulating the natural systems, bind with high affinity to G4 DNA and much lower affinity to duplex DNA. Notably, both enantiomers of a metallohelix containing a m-xylenyl bridge (5 b) were found to effectively inhibit primer elongation catalyzed by Taq DNA polymerase by stabilizing G4 structures formed in the template strands containing c-myc and c-kit2 G4-forming sequences. Moreover, both enantiomers of 5 b downregulated the expression of c-MYC and c-KIT oncogenes in human embryonic kidney cells at mRNA and protein levels. As metallohelices also bind alternative nucleic acid structures, they hold promise as potential multitargeted drugs.

Teresa B. Fitzpatrick.

Teresa B. Fitzpatrick.

De novo determination of near-surface electrostatic potentials by NMR

Electrostatic potentials computed from three-dimensional structures of biomolecules by solving the Poisson-Boltzmann equation are widely used in molecular biophysics, structural biology, and medicinal chemistry. Despite the approximate nature of the Poisson-Boltzmann theory, validation of the computed electrostatic potentials around biological macromolecules is rare and methodologically limited. Here, we present a unique and powerful NMR method that allows for straightforward and extensive comparison with electrostatic models for biomolecules and their complexes. This method utilizes paramagnetic relaxation enhancement arising from analogous cationic and anionic cosolutes whose spatial distributions around biological macromolecules reflect electrostatic potentials. We demonstrate that this NMR method enables de novo determination of near-surface electrostatic potentials for individual protein residues without using any structural information. We applied the method to ubiquitin and the Antp homeodomain-DNA complex. The experimental data agreed well with predictions from the Poisson-Boltzmann theory. Thus, our experimental results clearly support the validity of the theory for these systems. However, our experimental study also illuminates certain weaknesses of the Poisson-Boltzmann theory. For example, we found that the theory predicts stronger dependence of near-surface electrostatic potentials on ionic strength than observed in the experiments. Our data also suggest that conformational flexibility or structural uncertainties may cause large errors in theoretical predictions of electrostatic potentials, particularly for highly charged systems. This NMR-based method permits extensive assessment of near-surface electrostatic potentials for various regions around biological macromolecules and thereby may facilitate improvement of the computational approaches for electrostatic potentials.

NIR Light‐Triggered Shape Memory Polymers Based on Mussel‐Inspired Iron–Catechol Complexes

AbstractShape memory polymers (SMPs) with the permanent shape reconfiguration capability have received much research interest because they are capable of diversified tasks and the ability to work in conditions that required complex geometry. However, most of such SMPs are thermally triggered, which limits their applications. Inspired by reversible mussel adhesive protein chemistry, NIR light‐triggered SMPs with the permanent shape reconfiguration capability are prepared. The polymer networks are constructed using biocompatible polyethylene glycol, which is crosslinked based on catechol–Fe3+ coordination. The polymer networks have a uniform network structure and exhibit a considerable one‐way shape memory effect (1W‐SME) as well as a good two‐way shape memory effect (2W‐SME) under stress conditions. Taking advantage of the dynamic nature of the catechol–Fe3+ coordination, the permanent shape of the polymers could be reconfigured. Moreover, the catechol–Fe3+ complexes have a broad absorption in the NIR window, which bestows the polymers with excellent NIR light‐triggered SME. Further, the great potential of the obtained polymers in biomedical and electronic applications is presented. Owing to the NIR‐triggered 1W‐SME and the permanent shape reconfiguration capability, the polymer could be used as a personalizing vascular stent. Additionally, the polymer could be applied in light‐driven switches based on the NIR light‐triggered 2W‐SME.

Concise Review: How are Red Blood Cells Born, How do they Live and Die?

The average life cycle of a human RBC is approximately 120 days. Generally, by this point, the cell is worn out and damaged. RBCs pass through both the spleen and liver, where specialized immune cells called macrophages are found. Macrophages recognize when an RBC is spent, and undergo a process called phagocytosis where they digest the cell. In this process, the iron in hemoglobin is recycled for use in new blood cells and the hem molecule is degraded, conjugated to bilirubin, and eliminated from the body. All the other cellular proteins are either recycled or eliminated. Historically, this process was thought to occur exclusively in the spleen, but recent studies have shown that it occurs in the bone marrow .The RBC has been analyzed from many perspectives: cytological, hematological, and immunological, as well as from the focus of molecular biology, biophysics, and mathematics. Here we analyze how are red blood cells born and how they live and die in a brief overview of the whole process with special mention of the morphological aspects from bone marrow and spleen provided by Transmission and Scanning Electron Microscopy.

Liquid-liquid phase separation of tau: From molecular biophysics to physiology and disease.

Biomolecular condensation via liquid–liquid phase separation (LLPS) of intrinsically disordered proteins/regions (IDPs/IDRs), with and without nucleic acids, has drawn widespread interest due to the rapidly unfolding role of phase‐separated condensates in a diverse range of cellular functions and human diseases. Biomolecular condensates form via transient and multivalent intermolecular forces that sequester proteins and nucleic acids into liquid‐like membrane‐less compartments. However, aberrant phase transitions into gel‐like or solid‐like aggregates might play an important role in neurodegenerative and other diseases. Tau, a microtubule‐associated neuronal IDP, is involved in microtubule stabilization, regulates axonal outgrowth and transport in neurons. A growing body of evidence indicates that tau can accomplish some of its cellular activities via LLPS. However, liquid‐to‐solid transition resulting in the abnormal aggregation of tau is associated with neurodegenerative diseases. The physical chemistry of tau is crucial for governing its propensity for biomolecular condensation which is governed by various intermolecular and intramolecular interactions leading to simple one‐component and complex multi‐component condensates. In this review, we aim at capturing the current scientific state in unveiling the intriguing molecular mechanism of phase separation of tau. We particularly focus on the amalgamation of existing and emerging biophysical tools that offer unique spatiotemporal resolutions on a wide range of length‐ and time‐scales. We also discuss the link between quantitative biophysical measurements and novel biological insights into biomolecular condensation of tau. We believe that this account will provide a broad and multidisciplinary view of phase separation of tau and its association with physiology and disease.

Automated tangential-flow diafiltration device

Tangential flow filtration (TFF) is a chemical unit operation used to purify and concentrate liquid suspensions of colloids, proteins, or cells. The solution flows tangentially across a membrane, such that a selective part of the fluid permeates the membrane while the filtrated matter is retained, increasing its concentration. TFF is a mild mechanical purification method that does not interact chemically with the filtrate. It is applied in sensitive separation tasks in protein chemistry, microbiology, or immunology. It is a fast alternative for dialysis applications, also applicable in the field of colloid purification. However, the costs of automated lab-scale devices (30,000 €) and the consumable membrane modules (100–600 €) make TFF currently hardly accessible for lab-scale polymer researchers. Therefore, we built a low-cost TFF system (2400 €) partly automated by an Arduino microcontroller and optimized for diafiltration buffer exchange and concentration processes in soft matter colloid research. We use medical hemodialysis membrane modules that only cost a share (20–50 €) of alternative TFF modules, and we demonstrate the functionality of the system for an exemplary colloidal microgel purification process.

Buffers, Especially the Good Kind.

Fifty-five years ago, Norman Good and colleagues authored a paper that fundamentally advanced wet biochemistry [Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467-477] and in doing so has amassed more than 2500 citations. They laid out the properties required for useful, biochemically relevant hydrogen-ion buffers and then synthesized and tested 10 of them. Soon after, these buffers became commercially available. Since then, most of us never gave them a second thought. We just use them. Here, I discuss some of the background regarding the genesis of "Good's buffers", make a few (disparaging) observations about the non-Good's buffer, Tris, and suggest that we synthesize new buffers by combining the ideas of Good et al. with results from the past 60 years of protein chemistry.

Incorporating Molecular Biophysics in the Undergraduate Curriculum

The traditional introductory physics sequence doesn't work for life-science students. They don't find the traditional introductory topics - kinematics and Newton's laws to be relevant to their interests. That, combined with the well-known student misconceptions concerning describing motion mathematically, suggests that there's a need for a new starting point for the introductory physics sequence for life-science students. This presentation outlines a new active-learning approach that starts with the “marble game”, which simulates diffusion - a topic that students already know is fundamental to the life sciences. Students first learn how to play the game using a hands-on activity. They then implement the game in Excel producing a live graph of diffusion that leads them to discover that Fick's law of diffusion can be explained by unbiased random jumping between two boxes. In a guided-inquiry environment, students then apply the modeling techniques they learned from the marble game to drug elimination; radioactive decay; osmosis; ligand binding; enzyme kinetics; the Boltzmann factor; thermodynamics, phase equilibrium and entropy; random walks; membrane voltage, RC circuits and the action potential; models of the spread of COVID-19; Newtonian mechanics and biomechanics. The materials focus on engaging students in computational activities using kinetic Monte Carlo simulations and finite difference methods. These Excel activities allow them to discover for themselves the consequences and significance of model assumptions by reading and interpreting graphs, and by comparing model predictions with real data using linear regression and non-linear least-squares fits. A central theme of the materials is “thermodynamics from kinetics.” I've been successfully using this active-learning approach with undergraduate students for over 15 years. It provides a guided-inquiry environment that allows students to discover for themselves that science is an evidence-based endeavor with testable hypotheses that are supported by experiment. See http://circle4.com/biophysics for free textbook chapters and instructional videos.

Inside Cover: Synthetic Homogeneous Glycoforms of the SARS‐CoV‐2 Spike Receptor‐Binding Domain Reveals Different Binding Profiles of Monoclonal Antibodies (Angew. Chem. Int. Ed. 23/2021)

The receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein can be prepared by semi-synthesis as a homogeneous glycoform. In their Research Article on page 12904, Ping Wang and co-workers report the inaugural synthesis of glycosylated RBDs bearing structurally defined glycans. The cover picture illustrates the combination of carbohydrate and protein chemistry leading to the successful synthesis of this protein.

Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey.

As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.

High Potential for Biomass-Degrading Enzymes Revealed by Hot Spring Metagenomics.

Enzyme stability and activity at elevated temperatures are important aspects in biotechnological industries, such as the conversion of plant biomass into biofuels. In order to reduce the costs and increase the efficiency of biomass conversion, better enzymatic processing must be developed. Hot springs represent a treasure trove of underexplored microbiological and protein chemistry diversity. Herein, we conduct an exploratory study into the diversity of hot spring biomass-degrading potential. We describe the taxonomic diversity and carbohydrate active enzyme (CAZyme) coding potential in 71 publicly available metagenomic datasets from 58 globally distributed terrestrial geothermal features. Through taxonomic profiling, we detected a wide diversity of microbes unique to varying temperature and pH ranges. Biomass-degrading enzyme potential included all five classes of CAZymes and we described the presence or absence of genes encoding 19 glycosyl hydrolases hypothesized to be involved with cellulose, hemicellulose, and oligosaccharide degradation. Our results highlight hot springs as a promising system for the further discovery and development of thermo-stable biomass-degrading enzymes that can be applied toward generation of renewable biofuels. This study lays a foundation for future research to further investigate the functional diversity of hot spring biomass-degrading enzymes and their potential utility in biotechnological processing.

The Interaction of Human Glutathione Transferase GSTA1-1 with Reactive Dyes

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts.

Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.

Interaction standards for biophysics: anti-lysozyme nanobodies

There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb’s or ‘nanobodies’ against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards.

Subretinal Rather Than Intravitreal Adeno-Associated Virus-Mediated Delivery of a Complement Alternative Pathway Inhibitor Is Effective in a Mouse Model of RPE Damage.

PurposeThe risk for age-related macular degeneration has been tied to an overactive complement system. Despite combined attempts by academia and industry to develop therapeutics that modulate the complement response, particularly in the late geographic atrophy form of advanced AMD, to date, there is no effective treatment. We have previously demonstrated that pathology in the smoke-induced ocular pathology (SIOP) model, a model with similarities to dry AMD, is dependent on activation of the alternative complement pathway and that a novel complement activation site targeted inhibitor of the alternative pathway can be delivered to ocular tissues via an adeno-associated virus (AAV).MethodsTwo different viral vectors for specific tissue targeting were compared: AAV5-VMD2-CR2-fH for delivery to the retinal pigment epithelium (RPE) and AAV2YF-smCBA-CR2-fH for delivery to retinal ganglion cells (RGCs). Efficacy was tested in SIOP (6 months of passive smoke inhalation), assessing visual function (optokinetic responses), retinal structure (optical coherence tomography), and integrity of the RPE and Bruch's membrane (electron microscopy). Protein chemistry was used to assess complement activation, CR2-fH tissue distribution, and CR2-fH transport across the RPE.ResultsRPE- but not RGC-mediated secretion of CR2-fH was found to reduce SIOP and complement activation in RPE/choroid. Bioavailability of CR2-fH in RPE/choroid could be confirmed only after AAV5-VMD2-CR2-fH treatment, and inefficient, adenosine triphosphate–dependent transport of CR2-fH across the RPE was identified.ConclusionsOur results suggest that complement inhibition for AMD-like pathology is required basal to the RPE and argues in favor of AAV vector delivery to the RPE or outside the blood-retina barrier.

PPI-GA: A Novel Clustering Algorithm to Identify Protein Complexes within Protein-Protein Interaction Networks Using Genetic Algorithm

Comprehensive analysis of proteins to evaluate their genetic diversity, study their differences, and respond to the tensions is the main subject of an interdisciplinary field of study called proteomics. The main objective of the proteomics is to detect and quantify proteins and study their post-translational modifications and interactions using protein chemistry, bioinformatics, and biology. Any disturbance in proteins interactive network can act as a source for biological disorders and various diseases such as Alzheimer and cancer. Most current computational methods for discovering protein complexes are usually based on specific topological characteristics of protein-protein networks (PPI). To identify the protein complexes, in this paper, we, first, present a new encoding method to represent solutions; we then propose a new clustering algorithm based on the genetic algorithm, named PPI-GA, employing a new multiobjective quality function. The proposed algorithm is evaluated on two gold standard and real-world datasets. The result achieved demonstrates that the proposed algorithm can detect important protein complexes, and it provides more accurate results compared with state-of-the-art protein complex identification algorithms.

O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity.

A major role for the intracellular posttranslational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area have focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here, we use synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we find that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we find that although O-GlcNAc levels are globally reduced in Alzheimer’s disease brains, the modification of relevant sHSPs is either maintained or increased, suggesting a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

A Biochemical Perspective of the Nonstructural Proteins (NSPs) and the Spike Protein of SARS CoV-2.

The global pandemic that shut down the world in 2020 was caused by the virus, SARS CoV-2. The chemistry of the various nonstructural proteins (NSP3, NSP5, NSP12, NSP13, NSP14, NSP15, NSP16) of SARS CoV-2 is discussed. Secondly, a recent major focus of this pandemic is the variant strains of SARS CoV-2 that are increasingly occurring and more transmissible. One strain, called “D614G”, possesses a glycine (G) instead of an aspartate (D) at position 614 of the spike protein. Additionally, other emerging strains called “501Y.V1” and “501Y.V2” have several differences in the receptor binding domain of the spike protein (N501Y) as well as other locations. These structural changes may enhance the interaction between the spike protein and the ACE2 receptor of the host, increasing infectivity. The global pandemic caused by SARS CoV-2 is a rapidly evolving situation, emphasizing the importance of continuing the efforts to interrogate and understand this virus.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10930-021-09967-8.

Association Between rs12037447, rs146732504, rs151078858, rs55723436, and rs6094136 Polymorphisms and Kawasaki Disease in the Population of Polish Children.

Background: Kawasaki disease (KD) is an acute self-limited febrile vasculitis that mainly affects young children. Coronary artery involvement is the most serious complication in children with KD. It is currently the leading cause of acquired cardiac disease in children from developed countries. Literature data indicate a significant role of genetic susceptibility to KD.Objective: The aim of this study was to perform the first Genome-Wide Association Study (GWAS) in a population of Polish children with KD and identify susceptible genes involved in the pathogenesis of KD.Materials and Methods: The blood samples of Kawasaki disease patients (n = 119) were collected between 2016 and 2020, isolated and stored at the Department of Pediatrics, Nutrition and Metabolic Diseases, Children's Memorial Health Institute in Warsaw. The control group was based on Polish donors (n = 6,071) registered as the POPULOUS collection at the Biobank Lab of The Department of Molecular Biophysics in University of Lodz. DNA samples were genotyped for 558,231 Single Nucleotide Polymorphisms (SNPs) using the 24 × 1 Infinium HTS Human Core Exome microarrays according to the protocol provided by the manufacturer. In order to discover and verify genetic risk-factors for KD, association analysis was carried out using PLINK 1.9.Results: Of all 164,395 variants, 5 were shown to occur statistically (padjusted < 0.05) more frequent in Kawasaki disease patients than in controls. Those are: rs12037447 in non-coding sequence (padjusted = 8.329 × 10−4, OR = 8.697, 95% CI; 3.629–20.84) and rs146732504 in KIF25 (padjusted = 0.007354, OR = 11.42, 95% CI; 3.79–34.43), rs151078858 in PTPRJ (padjusted = 0.04513, OR = 8.116, 95% CI; 3.134–21.01), rs55723436 in SPECC1L (padjusted = 0.04596, OR = 5.596, 95% CI; 2.669–11.74), rs6094136 in RPN2 (padjusted = 0.04755, OR = 10.08, 95% CI; 3.385–30.01) genes.Conclusion: Polymorphisms of genes KIF25, PTRPJ, SPECC1L, RNP2 may be linked with the incidence of Kawasaki disease in Polish children.