Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Biophysical library screening using a Thermo-FMN assay to identify and characterize Clostridioides difficile FabK inhibitors.

Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism's fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme's FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.

Properties of phosphoramide benzoazole oligonucleotides (PABAOs). I. Structure and hybridization efficiency of N-benzimidazole derivatives.

In this work, we for the first time conducted a detailed study on the structure, dynamics, and hybridization properties of N-benzimidazole group-bearing phosphoramide benzoazole oligonucleotides (PABAOs) that we developed recently. By circular dichroism we established that the introduction of the modifications does not disrupt the B conformation of the DNA double helix. The formation of complexes is approximated by a two-state model. Complexes of PABAOs with native oligodeoxriboynucleotides form efficiently, and the introduction of such modifications reduces thermal stability of short duplexes (8-10 bp) by ∼5°Ð¡ per modification. Using UV-spectroscopy analysis, a neutral charge of the phosphate residue modified by the N-benzimidazole moiety in the pH range of 3-9.5 was found. The results confirm possible usefulness of PABAOs for both basic research and biomedical applications.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

PD-L1 DNA aptamers isolated from agarose-bead SELEX.

Increased expression and activity of the PD-L1/PD-1 pathway suppresses the activation of cytotoxic T cells, which is vital in anti-tumour defence, allowing tumours to rise, expand and progress. Current strategies using antibodies to target PD-1/PD-L1 have been very effective in cancer therapeutics and companion diagnostics. Aptamers are a new class of molecules that offer an alternative to antibodies. Herein, the systematic evolution of ligands by exponential enrichment (SELEX) using agarose slurry beads was conducted to isolate DNA aptamers specific to recombinant human PD-L1 (rhPD-L1). Isolated aptamers were sequenced and analysed using MEGA X and structural features were examined using mFold. Three aptamer candidates (P33, P32, and P12) were selected for evaluation of binding affinity (dissociation constant, Kd) using ELONA and specificity and competitive inhibition assessment using the potentiostat-electrochemical method. Among those three, P32 displayed the highest specificity (8 nM) against PD-L1. However, P32 competes for the same binding site with the control antibody, 28-8. This study warrants further assessment of P32 aptamer as a potential, cost-effective alternative tool for targeting PD-L1.

Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

Cyclodextrin-Enabled Enantioselective Complexation Study of Cathinone Analogs.

The characteristic alkaloid component of the leaves of the catnip shrub (Catha edulis) is cathinone, and its synthetic analogs form a major group of recreational drugs. Cathinone derivatives are chiral compounds. In the literature, several chiral methods using cyclodextrins (CDs) have been achieved so far for diverse sets of analogs; however, a comprehensive investigation of the stability of their CD complexes has not been performed yet. To characterize the enantioselective complex formation, a systematic experimental design was developed in which a total number of 40 neutral, positively, and negatively charged CD derivatives were screened by affinity capillary electrophoresis and compared according to their cavity size, substituent type, and location. The functional groups responsible for the favorable interactions were identified in the case of para-substituted cathinone analog mephedrone, flephedrone, and 4-methylethcathinone (4-MEC) and in the case of 3,4-methylendioxy derivative butylone and methylenedioxypyrovalerone (MDPV). The succinylated-ß-CD and subetadex exhibited the highest complex stabilities among the studied drugs. The complex stoichiometry was determined using the Job's plot method, and the complex structures were further studied using ROESY NMR measurements. The results of our enantioselective complex formation study can facilitate chiral method development and may lead to evaluate potential CD-based antidotes for cathinone analogs.

A novel consensus-based computational pipeline for screening of antibody therapeutics for efficacy against SARS-CoV-2 variants of concern including Omicron variant.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein's receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility, and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with 3-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero- dimeric and trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concerns (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant's RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118)-that may likely neutralize the recently emerged Omicron variant-facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.

RNA double strand hybridization measured at the single molecule level.

RNA double strand hybridization is a hallmark for gene expression regulation. In this function, single stranded regulatory RNA forms Watson-Crick base pairs with complementary messenger RNA. In the presented work the dissociation constants of complementary equally sized RNA single strands were measured at the single molecule level applying fluorescence correlation spectroscopy (FCS). Dissociation constants of 3.2 nM, 1.4 nM and 1.0 nM were determined for 26 bp, 41 bp and 54 bp dsRNA, respectively. The translational diffusion coefficients of RNA, measured at infinite dilution, could be accurately predicted applying the model D = 4.58 × 10-10 N-0.39 m2s-1.

Sequence-Dependent Interaction of the Human Papillomavirus E2 Protein with the DNA Elements on Its DNA Replication Origin.

The human papillomavirus (HPV) E2 protein is essential for regulating the initiation of viral DNA replication as well as the regulation of transcription of certain HPV-encoded genes. Its ability to recognize and bind to its four recognition sequences in the viral origin is a key step in the initiation of HPV DNA replication. Thus, understanding the mechanism of DNA binding by E2 protein and the unique roles played by individual DNA sequence elements of the replication origin is essential. We have purified the recombinant full-length HPV type 11 E2 protein. Quantitative DNA binding analysis indicated E2 protein bound all four DNA binding sites with reasonably high affinities but with distinct preferences. It bound its cognate binding sites 1, 2, and 4 with higher affinities, but bound binding site 3 with lower affinity. Analysis of binding to these sites unraveled multiple sequence elements that appeared to influence E2 binding affinity and target discrimination, including the sequence of spacer region, flanking sequences, and proximity of E2 binding sites. Thermodynamic analysis indicated hydrophobic interaction in the protein-DNA complex formation. Our studies indicate a large multi-protein complex formation on the HPV-origin DNA, likely due to reasonably high binding affinities as well as intrinsic oligomerization propensity of E2 dimers.

Microfluidic Diffusion Sizing Applied to the Study of Natural Products and Extracts That Modulate the SARS-CoV-2 Spike RBD/ACE2 Interaction.

The interaction between SARS-CoV-2 spike RBD and ACE2 proteins is a crucial step for host cell infection by the virus. Without it, the entire virion entrance mechanism is compromised. The aim of this study was to evaluate the capacity of various natural product classes, including flavonoids, anthraquinones, saponins, ivermectin, chloroquine, and erythromycin, to modulate this interaction. To accomplish this, we applied a recently developed a microfluidic diffusional sizing (MDS) technique that allows us to probe protein-protein interactions via measurements of the hydrodynamic radius (Rh) and dissociation constant (KD); the evolution of Rh is monitored in the presence of increasing concentrations of the partner protein (ACE2); and the KD is determined through a binding curve experimental design. In a second time, with the protein partners present in equimolar amounts, the Rh of the protein complex was measured in the presence of different natural products. Five of the nine natural products/extracts tested were found to modulate the formation of the protein complex. A methanol extract of Chenopodium quinoa Willd bitter seed husks (50 µg/mL; bisdesmoside saponins) and the flavonoid naringenin (1 µM) were particularly effective. This rapid selection of effective modulators will allow us to better understand agents that may prevent SARS-CoV-2 infection.

FFP: joint Fast Fourier transform and fractal dimension in amino acid property-aware phylogenetic analysis.

BACKGROUND: Amino acid property-aware phylogenetic analysis (APPA) refers to the phylogenetic analysis method based on amino acid property encoding, which is used for understanding and inferring evolutionary relationships between species from the molecular perspective. Fast Fourier transform (FFT) and Higuchi's fractal dimension (HFD) have excellent performance in describing sequences' structural and complexity information for APPA. However, with the exponential growth of protein sequence data, it is very important to develop a reliable APPA method for protein sequence analysis. RESULTS: Consequently, we propose a new method named FFP, it joints FFT and HFD. Firstly, FFP is used to encode protein sequences on the basis of the important physicochemical properties of amino acids, the dissociation constant, which determines acidity and basicity of protein molecules. Secondly, FFT and HFD are used to generate the feature vectors of encoded sequences, whereafter, the distance matrix is calculated from the cosine function, which describes the degree of similarity between species. The smaller the distance between them, the more similar they are. Finally, the phylogenetic tree is constructed. When FFP is tested for phylogenetic analysis on four groups of protein sequences, the results are obviously better than other comparisons, with the highest accuracy up to more than 97%. CONCLUSION: FFP has higher accuracy in APPA and multi-sequence alignment. It also can measure the protein sequence similarity effectively. And it is hoped to play a role in APPA's related research.

PPI-Affinity: A Web Tool for the Prediction and Optimization of Protein-Peptide and Protein-Protein Binding Affinity.

Virtual screening of protein-protein and protein-peptide interactions is a challenging task that directly impacts the processes of hit identification and hit-to-lead optimization in drug design projects involving peptide-based pharmaceuticals. Although several screening tools designed to predict the binding affinity of protein-protein complexes have been proposed, methods specifically developed to predict protein-peptide binding affinity are comparatively scarce. Frequently, predictors trained to score the affinity of small molecules are used for peptides indistinctively, despite the larger complexity and heterogeneity of interactions rendered by peptide binders. To address this issue, we introduce PPI-Affinity, a tool that leverages support vector machine (SVM) predictors of binding affinity to screen datasets of protein-protein and protein-peptide complexes, as well as to generate and rank mutants of a given structure. The performance of the SVM models was assessed on four benchmark datasets, which include protein-protein and protein-peptide binding affinity data. In addition, we evaluated our model on a set of mutants of EPI-X4, an endogenous peptide inhibitor of the chemokine receptor CXCR4, and on complexes of the serine proteases HTRA1 and HTRA3 with peptides. PPI-Affinity is freely accessible at https://protdcal.zmb.uni-due.de/PPIAffinity.

The measurement of binding affinities by NMR chemical shift perturbation.

We have carried out chemical shift perturbation titrations on three contrasting proteins. The resulting chemical shifts have been analysed to determine the best way to fit the data, and it is concluded that a simultaneous fitting of all raw shift data to a single dissociation constant is both the most accurate and the most precise method. It is shown that the optimal weighting of 15N chemical shifts to 1H chemical shifts is protein dependent, but is around the consensus value of 0.14. We show that chemical shift changes of individual residues can be fit to give residue-specific affinities. Residues with affinities significantly stronger than average are found in close contact with the ligand and are suggested to form a rigid contact surface, but only when the binding involves little conformational change. This observation may be of value in analysing binding and conformational change.

Detection of electrostatic molecular binding using the water proton signal.

PURPOSE: Saturation transfer MRI has previously been used to probe molecular binding interactions with signal enhancement via the water signal. Here, we detail the relayed nuclear overhauser effect (rNOE) based mechanisms of this signal enhancement, develop a strategy of quantifying molecular binding affinity, i.e., the dissociation constant ( KD$$ {K}_D $$ ), and apply the method to detect electrostatic binding of several charged small biomolecules. Another goal was to estimate the detection limit for transient receptor-substrate binding. THEORY AND METHODS: The signal enhancement mechanism was quantitatively described by a three-step magnetization transfer model, and numerical simulations were performed to verify this theory. The binding equilibria of arginine, choline, and acetyl-choline to anionic resin were studied as a function of ligand concentration, pH, and salt content. Equilibrium dissociation constants ( KD$$ {K}_D $$ ) were determined by fitting the multiple concentration data. RESULTS: The numerical simulations indicate that the signal enhancement is sufficient to detect the molecular binding of sub-millimolar (∼100 µM) concentration ligands to low micromolar levels of molecular targets. The measured rNOE signals from arginine, choline, and acetyl-choline binding experiments show that several magnetization transfer pathways (intra-ligand rNOEs and intermolecular rNOEs) can contribute. The rNOEs that arise from molecular ionic binding were influenced by pH and salt concentration. The molecular binding strengths in terms of KD$$ {K}_{\mathrm{D}} $$ ranged from 70-160 mM for the three cations studied. CONCLUSION: The capability to use MRI to detect the transient binding of small substrates paves a pathway towards the detection of micromolar level receptor-substrate binding in vivo.

New bis- and tetrakis-1,2,3-triazole derivatives: Synthesis, DNA cleavage, molecular docking, antimicrobial, antioxidant activity and acid dissociation constants.

In this study, a series of bis- and tetrakis-1,2,3-triazole derivatives were synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry in 73-95% yield. The bis- and tetrakis-1,2,3-triazoles exhibited significant DNA cleavage activity while the tetrakis-1,2,3-triazole analog 6g completely degraded the plasmid DNA. Molecular docking simulations suggest that compound 6g acts as minor groove binder of DNA by binding through several noncovalent interactions with base pairs. All bis- and tetrakis-1,2,3-triazole derivatives were screened for antibacterial activity against E. coli, B. cereus, S. aureus, P. aeruginosa, E. hirae, L. pneumophila subsp. pneumophila strains and antifungal activity against microfungus C. albicans and C. tropicalis strains. Compound 4d exhibited the best antibacterial activity among bis-1,2,3-triazoles against E. coli and E. hirae, while 6c exhibited the best antibacterial activity among tetrakis-1,2,3-triazoles against E. hirae. Furthermore, the best antifungal activity against C. albicans and C. tropicalis was reported for the compound 5, while 6d displayed the best antifungal activity against C. tropicalis and C. albicans. Reasonable iron chelating activities and DPPH radical scavenging abilities were found for some of the compounds. Finally, the acid dissociation constants (pKa) of the bis-1,2,3-triazoles were also determined with the help of HYPERQUAD program using the data obtained from potentiometric titrations. The reported data here concludes that the bis- and tetrakis-1,2,3-triazoles are important cores that should be considered for further development of especially new anticancer agents acting through the DNA cleavage activity.

A novel structure-based approach for identification of vertebrate susceptibility to SARS-CoV-2: Implications for future surveillance programmes.

Understanding the origin of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a highly debatable and unresolved issue for scientific communities all over the world. Understanding the mechanism of virus entry to the host cells is crucial to deciphering the susceptibility profiles of animal species to SARS-CoV-2. The interaction of SARS-CoV-2 ligands (receptor-binding domain on spike protein) with its host cell receptor, angiotensin-converting enzyme 2 (ACE2), is a critical determinant of host range and cross-species transmission. In this study, we developed and implemented a rigorous computational approach for predicting binding affinity between 299 ACE2 orthologs from diverse vertebrate species and the SARS-CoV-2 spike protein. The findings show that the SARS-CoV-2 spike protein can bind to a wide range of vertebrate species carrying evolutionary divergent ACE2, implying a broad host range at the virus entry level, which may contribute to cross-species transmission and further viral evolution. Furthermore, the current study facilitated the identification of genetic determinants that may differentiate susceptible from resistant host species based on the conservation of ACE2-spike protein interacting residues in vertebrate host species known to facilitate SARS-CoV-2 infection; however, these genetic determinants warrant in vivo experimental confirmation. The molecular interactions associated with varied binding affinity of distinct ACE2 isoforms in a specific bat species were identified using protein structure analysis, implying the existence of diversified bat species' susceptibility to SARS-CoV-2. The current study's findings highlight the importance of intensive surveillance programmes aimed at identifying susceptible hosts, especially those with the potential to transmit zoonotic pathogens, in order to prevent future outbreaks.

Capillary electrophoresis applied for the determination of acidity constants and limiting electrophoretic mobilities of ionizable herbicides including glyphosate and its metabolites and for their simultaneous separation.

Thermodynamic acidity constants and limiting ionic mobilities were determined for polyprotic non-chromophore analytes using capillary electrophoresis with capacitively coupled contactless conductivity detection. It was not necessary to work with buffers of identical ionic strength as ionic strength effects on effective electrophoretic mobilities were corrected by modeling during data evaluation (software AnglerFish). The mobility data from capillary electrophoresis coupled to conductivity detection were determined in the pH range from 1.25 to 12.02 with a high resolution (36 pH steps). With this strategy, thermodynamic acidity constants and limiting ionic mobilities for various acidic herbicides were determined, sometimes for the first time. The model analytes included glyphosate, its metabolites, and its acetylated derivates (aminomethyl phosphonic acid, glyoxylic acid, sarcosine, glycine, N-acetyl glyphosate, N-acetyl aminomethyl phosphonic acid, hydroxymethyl phosphonic acid). The obtained data were used in simulations to optimize separations by capillary electrophoresis. Simulations correlated very well to experimental results. With the new method, the separation of glyphosate from interfering components like phosphate in beer samples was possible.

Principles and practice of determining metal-protein affinities.

Metal ions play many critical roles in biology, as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissociation constant, KD, describes the thermodynamic strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to determine protein affinities for essential biological transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent experimental design can be applied to determine accurate KD values.

Fluorescence Lifetime Phasor Analysis of the Decamer-Dimer Equilibrium of Human Peroxiredoxin 1.

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (KD) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained KD (1.1 × 10-24 M4) and C0.5 (1.36 µM) values for the decamer-dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist's toolkit.