Equilibrium Titration Research Articles

IgY Antibodies from Birds: A Review on Affinity and Avidity.

IgY antibodies are found in the blood and yolk of eggs. Several studies show the feasibility of utilising IgY for immunotherapy and immunodiagnosis. These antibodies have been studied because they fulfil the current needs for reducing, replacing, and improving the use of animals. Affinity and avidity represent the strength of the antigen-antibody interaction and directly influence antibody action. The aim of this review was to examine the factors that influence the affinity and avidity of IgY antibodies and the methodologies used to determine these variables. In birds, there are few studies on the maturation of antibody affinity and avidity, and these studies suggest that the use of an adjuvant-type of antigen, the animal lineage, the number of immunisations, and the time interfered with the affinity and avidity of IgY antibodies. Regarding the methodologies, most studies use chaotropic agents to determine the avidity index. Studies involving the solution phase and equilibrium titration reactions are also described. These results demonstrate the need for the standardisation of methodologies for the determination of affinity and avidity so that further studies can be performed to optimise the production of high avidity IgY antibodies.

Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

Method for direct measuring antibody affinity in biological sample.

We are reporting a new method to measure the affinity of specific antibodies in biological sample without the need of purification, or direct labeling. It was used to measure the affinity of anti-RBD (SARS-CoV2) IgG in plasma from Covid-19 recovered patient and vaccinated populations. Among the vaccinated population, we also measured the affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-Cov-2 virus and the Omicron variants. The method has provided a quantitative way to evaluate antibodies produced by vaccination. The new method involves a classical Solution phase Equilibrium Titration step and an Imaging based Detection step (SET-ID). In the Solution phase Titration step, the antibody was kept at constant concentration (lower than the expected KD value) and mixed with various concentrations of antigen solution; In the Imaging Detection step, the amount of free antibody (not bound to antigen) at each antigen concentration was probed with small amount of microparticles coated with antigen and then the labeled secondary antibody. In this presentation, we will discuss the critical experimental condition, validation, and limitations. For verification, the binding curves of an antibody were compared using a well-established fluorescence spectroscopic-based method and the SET-ID method. The incubation time and the number of microparticles used in the second step were evaluated experimentally and theoretically to ensure minimal perturbation to the solution phase equilibrium, while the fraction of free antibody presented in the solution can still be measured by the signal on the microparticles.

Exploring The Promiscuity Potential Of 6‐Hydroxynicotinate‐3‐Monooxygenase: Consequences To Catalysis Of Adding A Nitrogen At C5 Within The Substrate’s Aromatic Ring

Bacterial degradation of nicotinic acid is a model for exploring the bioremediation potential of soil bacteria. One enzyme within this catabolic pathway is 6‐hydroxynicotinate‐3‐monooxygenase (NicC), a group A flavoenzyme that catalyzes the conversion of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine. This decarboxylative hydroxylation reaction is relatively unique in biology. We recently published a study highlighting NicC’s ability to catalyze the decarboxylation and hydroxylation reaction with alternative substrates including 4‐hydroxybenzoate (4HBA) and 5‐chloro‐6‐hydroxynicotinate (5‐Cl‐6HNA). To further explore the structural basis of a substrate that enables tight binding and catalysis with NicC, 2‐hydroxypyrimidine‐5‐caboxylate (2HM5A) was characterized. This 6‐HNA analogue with an extra N atom at C5 was chosen due to its fundamental symmetry and comparable electron distribution as 5‐Cl‐6HNA. Analyses of NicC with 2HM5A show that the expected product, 2,5‐dihydroxypyrimidine (isouracil), is generated, with H2O2 occurring at a mol fraction of about 0.07, indicating that uncoupling between NADH oxidation and substrate hydroxylation is minimal. Equilibrium titration of NicC with 2HM5A by changes in A‐450 nm measure the Kd = 39 ± 6 µM, indicating that this substrate is bound more tightly than 6‐HNA (Kd= 58 ± 12 µM) but less tightly than 5‐Cl‐6‐HNA (Kd= 7 ± 2 µM). Interestingly, the overall ΔA450 elicited by the binding of 2HM5A (0.018) was smaller in comparison to the ΔA450 observed for the binding of 5‐Cl‐6HNA (0.07) or 6‐HNA (0.05). Attenuated flavin movement due the extra nitrogen of the pyrimidine ring in 2HM5A H‐bonding to the isoalloxazine ring may explain this observation. The kinetics of binding 2HM5A by NicC measured by stopped flow spectrophotometry shows that this analogue binds by a two‐step mechanism, with a nearly ten‐fold higher initial affinity for NicC prior to isomerization (ionization) than 6‐HNA. This added affinity may again be explainable by the added H‐bonding potential of this analogue. In the second step of binding, 2HM5A was observed to have a rate constant similar to 5‐Cl‐6HNA (k2= 220 s‐1), possibly indicating that increased electron density at/near the C5 position accelerates this phase of the binding mechanism. How 2HM5A affects NicC’s mechanism of flavin reduction and oxidation will also be presented. These findings provide further evidence of this monooxygenase’s potential to be engineered to hydroxylate other N‐heterocyclic aromatic compounds in bioremediation.

Novel CALM3 Variant Causing Calmodulinopathy With Variable Expressivity in a 4-Generation Family.

CaM (calmodulin), encoded by 3 separate genes (CALM1, CALM2, and CALM3), is a multifunctional Ca2+-binding protein involved in many signal transduction events including ion channel regulation. CaM variants may present with early-onset long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia, or sudden cardiac death. Most reported variants occurred de novo. We identified a novel CALM3 variant, p.Asn138Lys (N138K), in a 4-generation family segregating with LQTS. The aim of this study was to elucidate its pathogenicity and to compare it with that of p.D130G-CaM-a variant associated with a severe LQTS phenotype. We performed whole exome sequencing for a large, 4-generation family affected by LQTS. To assess the effect of the detected CALM3 variant, the intrinsic Ca2+-binding affinity was measured by stoichiometric Ca2+ titrations and equilibrium titrations. L-type Ca2+ and slow delayed rectifier potassium currents (ICaL and IKs) were recorded by whole-cell patch-clamp. Cav1.2 and Kv7.1 membrane expression were determined by optical fluorescence assays. We identified 14 p.N138K-CaM carriers in a family where 2 sudden deaths occurred in children. Several members were only mildly affected compared with CaM-LQTS patients to date described in literature. The intrinsic Ca2+-binding affinity of the CaM C-terminal domain was 10-fold lower for p.N138K-CaM compared with wild-type-CaM. ICaL inactivation was slowed in cells expressing p.N138K-CaM but less than in p.D130G-CaM cells. Unexpectedly, a larger IKs current density was observed in cells expressing p.N138K-CaM, but not for p.D130G-CaM, compared with wild-type-CaM. The p.N138K CALM3 variant impairs Ca2+-binding affinity of CaM and ICaL inactivation but potentiates IKs. The variably expressed phenotype of this variant compared with previously published de novo LQTS-CaM variants is likely explained by a milder impairment of ICaL inactivation combined with IKs augmentation.

Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics.

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

Photogeneration and reactivity of flavin anionic semiquinone in a bifurcating electron transfer flavoprotein

Electron transfer bifurcation allows production of a strongly reducing carrier at the expense of a weaker one, by redistributing energy among a pair of electrons. Thus, two weakly-reducing electrons from NADH are consumed to produce a strongly reducing ferredoxin or flavodoxin, paid for by reduction of an oxidizing acceptor. The prevailing mechanism calls for participation of a strongly reducing flavin semiquinone which has been difficult to observe with site-certainly in multi-flavin systems. Using blue light (450 nm) to photoexcite the flavins of bifurcating electron transfer flavoprotein (ETF), we demonstrate accumulation of anionic flavin semiquinone in excess of what is observed in equilibrium titrations, and establish its ability to reduce the low-potential electron acceptor benzyl viologen. This must occur at the bifurcating flavin because the midpoint potentials of the electron transfer (ET) flavin are not sufficiently negative. We show that bis-tris propane buffer is an effective electron donor to the flavin photoreduction, but that if the system is prepared with the ET flavin chemically reduced, so that only the bifurcating flavin is oxidized and photochemically active, flavin anionic semiquinone is formed more rapidly. Thus, excited bifurcating flavin is able to draw on an electron stored at the ET flavin. Flavin semiquinone photogenerated at the bifurcation site must therefore be accompanied by additional semiquinone formation by oxidation of the ET flavin. Consistent with the expected instability of bifurcating flavin semiquinone, it subsides immediately upon cessation of illumination. However comparison with yields of semiquinone in equilibrium titrations suggest that during continuous illumination at pH 9 a steady state population of 0.3 equivalents of bifurcating flavin semiquinone accumulates, and then undergoes further photoreduction to the hydroquinone. Although transient, the population of bifurcating flavin semiquinone explains the system's ability to conduct light-driven electron transfer from bis-tris propane to benzyl viologen, in effect trapping energy from light.

The Effect of Salt on the Interactions Between Droplets in Water-in-Oil Microemulsions

The interaction enthalpies, entropies and Gibbs energies between microemulsion droplets for {water/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-decane}, {water/NaCl/AOT/n-decane} and {water/CaCl2/AOT/n-decane}, with the molar ratio ω of water to AOT being 26.3 and the concentration of salt in water being 0.02 mol·L–1, have been investigated by measurements of liquid–liquid phase equilibrium and isothermal titration microcalorimetry. The interaction enthalpies determined from isothermal titration microcalorimetry and liquid–liquid equilibrium are consistent. The interaction was found to be enhanced as the ionic strength increases. From the data of liquid–liquid equilibriums collected in the critical region, the critical exponent β corresponding to the width of the coexistence curve was determined, which showed good agreement with the 3D-Ising value and supported the pseudo binary solution proposal for these multiple microemulsions. A thermodynamic approach based on the Carnahan–Starling–van der Waals type equation was improved and used to deduce the interaction properties between droplets in the microemulsions. Salt effects on the microemusion phase equilibria are also discussed.

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32 P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff /kon determines Kd . However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equilibrium binding regime. We also observed effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.

Short-lived neutral FMN and FAD semiquinones are transient intermediates in cryo-reduced yeast NADPH-cytochrome P450 reductase

The electron configuration of flavin cofactors, FMN and FAD, is a critical factor governing the reactivity of NADPH-cytochrome P450 reductase (CPR). The current view of electron transfer by the mammalian CPR, based on equilibrium redox potentials of the flavin cofactors, is that the two electron-reduced FMN hydroquinone (FMNH2), rather than one electron-reduced FMN semiquinone, serves as electron donor to the terminal protein acceptors. However, kinetic and thermodynamic studies on the CPR species originated from different organisms have shown that redox potentials measured at distinct electron transfer steps differ from redox potentials determined by equilibrium titration. Collectively, previous observations suggest that the short-lived transient semiquinone species may carry electrons in diflavin reductases. In this work, we have investigated spectroscopic properties of the CPR-bound FAD and FMN reduced at 77 K by radiolytically-generated thermalized electrons. Using UV–vis spectroscopy, we demonstrated that upon cryo-reduction of oxidized yeast CPR (yCPR) containing an equimolar ratio of both FAD and FMN, or FAD alone, neutral semiquinones were trapped at 77 K. During annealing at the elevated temperatures, unstable short-lived neutral semiquinones relaxed to spectroscopically distinct air-stable neutral semiquinones. This transition was independent of pH within the 6.0–10.7 range. Our data on yeast CPR are in line with the previous observations of others that the flavin short-lived transient semiquinone intermediates may have a role in the electron transfer by CPR at physiological conditions.

Interplay of actin, ADP and Mg2+ interactions with striated muscle myosin: Implications of their roles in ATPase

The effects of Mg2+ on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg2+ inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg2+ were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg2+ bound to a fluorescent ADP analog about five-times tighter than without Mg2+. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg2+. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (Kd) without Mg2+ was more than five-fold smaller than with Mg2+. Further, we examined effects of F-actin, ADP and Mg2+ binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg2+-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg2+ affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.

Affinity determination of biomolecules: a kinetic model for the analysis of pre-equilibrium titration curves.

Equilibrium titration curves have been used to determine the affinity of biomolecules using a variety of platforms and technologies. Such measurements, however, are experimentally limited by the time that is needed for the system to reach the equilibrium, which may exceed the time during which the studied biomolecules are stable. We propose here a kinetic model to analyze titration curves obtained prior to full equilibration of the system.

Revealing the distinct folding phases of an RNA three-helix junction.

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

An Exact Model of Daptomycin Binding to Lipid Bilayers: An Update

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions. A preliminary version of the model was presented at the Biophysical Society Meeting in 2017. Here, we present a more complete analysis.

Dissecting the Molecular Mechanism for Familial Cardiomyopathies

The heart needs to generate sufficient force and power to pump blood to the body and to adjust its power output in response to stresses. Mutations of contractile proteins, such as cardiac troponin T, can lead to familial cardiomyopathies, the leading cause of sudden cardiac death in people under the age of 30. Two common forms of cardiomyopathy, hypertrophic (HCM) and dilated (DCM) are characterized by thickening or thinning of the ventricular wall, respectively. However, how changes in contractile proteins at the molecular level lead to the disease phenotype is not well understood. To address this problem, we examined two point mutations in human cardiac troponin T (TnT), R92Q and ΔK210, which cause hypertrophic and dilated cardiomyopathy, respectively, in human patients. We have reconstituted the calcium-based regulation of actomyosin in vitro using tissue purified and recombinant cardiac proteins. Similar to previous studies, in vitro motility assays which reconstitute the calcium-dependent interactions between myosin and regulated thin filaments revealed that R92Q is shifted towards submaximal calcium activation while ΔK210 is shifted towards supermaximal calcium activation. We dissected the molecular mechanism of these differential changes using a combination of fluorescence equilibrium titrations and stopped-flow experiments, and show that these effects are due to alterations in the free energies of the biochemical transitions that regulate muscle activation. In particular, we see that these mutations differentially affect calcium-dependent positioning of tropomyosin along the thin filament. We propose that these changes affect the regulation of force production by the molecular motor myosin, leading to alterations in mechanosensing by heart tissue.

Supersaturated calcium carbonate solutions are classical.

Mechanisms of CaCO3 nucleation from solutions that depend on multistage pathways and the existence of species far more complex than simple ions or ion pairs have recently been proposed. Herein, we provide a tightly coupled theoretical and experimental study on the pathways that precede the initial stages of CaCO3 nucleation. Starting from molecular simulations, we succeed in correctly predicting bulk thermodynamic quantities and experimental data, including equilibrium constants, titration curves, and detailed x-ray absorption spectra taken from the supersaturated CaCO3 solutions. The picture that emerges is in complete agreement with classical views of cluster populations in which ions and ion pairs dominate, with the concomitant free energy landscapes following classical nucleation theory.

Investigation on the Interaction of Pseudomonas syringae Effector AvrPto with AtRabE1d GTPase.

The tomato bacterial speck is a worldwide disease. It is caused by the infection of pathogenic Pseudomonas syringae pv. tomato which delivers the effector AvrPto into the host cells via the type III secretion system. AvrPto interacts with a Rab8 subfamily protein in the GTP-bound form and participates in the response to pathogen infection, but the pathogenic mechanism involved remains elusive. The main objective of this study was to investigate on the interrelationship of AvrPto with AtRabE1d and Pto, which would allow us to have a deeper understanding of the pathogen mechanism of bacterial speck mediated by avirulent AvrPto and provides theoretic support for the prevention and cure of tomato bacterial speck disease. AvrPto8-159 and AtRabE1d13-185Q74L proteins were expressed in Escherichia coli expression system, purified via nickel affinity chromatography and size exclusion chromatography, and identified by SDS-PAGE. The interaction of AvrPto8-159 with AtRabE1d13-185Q74L was confirmed in vitro based on the fluorescence resonance energy transfer. In addition, the affinity of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined using the fluorescence polarization based equilibrium titration. The comparison of the complex structural model of AtRabE1d with AvrPto, which was docked and refined by Patchdock and FireDock softwares. AvrPto8-159 and AtRabE1d13-185Q74L can be expressed and purified well in E.coli. FRET between AvrPto8-159 with GFP-tag and mantGTP-load AtRabE1d13-185Q74L was observed slightly in vitro for the first time. On the other hand, deploying DsRed of AtRabE1d13-185Q74L as FRET partner with GFP, AvrPto was shown to interact with AtRabE1d more significantly with increasing concentrations of DsRed-AtRabE1d13-185Q74L. The equilibrium dissociation constant of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined to be 13.5 μM. This work reports preparation and interaction of AvrPto8-159 with AtRabE1d13- 185Q74L. AvrPto8-159 exhibited medium affinity with AtRabE1d13-185Q74L based on the fluorescence polarization. From the structural model of the complex AvrPto:AtRabE1d, AvrPto interacted with AtRabE1d and Pto proteins with completely different biding sites.

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m-1 s-1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein.

An Exact Model of Daptomycin Binding to Lipid Bilayers

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions.

The Role of Intrinsically Disordered Linker and Salt Type on Cooperativity of E. Coli SSB Binding to ssDNA

E. coli single strand DNA binding protein (SSB) is a key protein in DNA replication, recombination and repair. SSB functions as a homotetramer and binds ssDNA in different modes that differ in their excluded site size and cooperative binding to ssDNA. Two types of cooperativity have been identified previously: the high “unlimited”, nearest-neighbor type of cooperativity associated with the 35 site size ((SSB)35) binding mode at low [NaCl], and a lower, “limited” cooperativity associated with formation of dimers of tetramers in the 65 site size ((SSB)65) binding mode at [NaCl] > 200 mM. Using sedimentation velocity and equilibrium titration methods we have examined how salt concentration and type affects these cooperativities and binding mode transitions on long ssDNA, focusing on the effects of KGlu, since glutamate is the major monovalent anion in E. coli. Unexpectedly, we find that in contrast to NaCl, KGlu promotes highly cooperative binding to ssDNA even at high concentrations of 0.5 M, conditions that promote the ((SSB)65) mode, suggesting that highly cooperative binding is likely occurring in vivo. We also show that all cooperative binding behavior absolutely requires the unstructured C-terminal region (intrinsically disordered linker (IDL)) connecting the SSB DNA binding core and the 9 amino acid C-terminal end (tip) needed for SSB binding to other proteins involved in genome maintenance (supported by NIH GM030498 to TML).