Allosteric effects of E. coli SSB and RecR proteins on RecO protein binding to DNA.

Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO-ssDNA aggregates occur that form with higher propensity on ssDNA of increasing length. Binding of RecO to the SSB-Ct peptide inhibits RecO-ssDNA aggregation. RecOR complexes can bind ssDNA via RecO, but aggregation is suppressed even in the absence of the SSB-Ct peptide, demonstrating an allosteric effect of RecR on RecO binding to ssDNA. Under conditions where RecO binds ssDNA but does not form aggregates, SSB-Ct binding enhances the affinity of RecO for ssDNA. For RecOR complexes bound to ssDNA, we also observe a shift in RecOR complex equilibrium towards a RecR4O complex upon binding SSB-Ct. These results suggest a mechanism by which SSB recruits RecOR to facilitate loading of RecA onto ssDNA gaps.

Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations.

Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions. Here we present a global multi-method analysis that synergistically bridges gaps in resolution and sensitivity of orthogonal techniques. We demonstrate the method with a panel of monoclonal antibodies exhibiting different degrees of self-association. We show how their concentration-dependent behavior, examined by static and dynamic light scattering and sedimentation velocity, can be jointly described in a self-consistent framework that separates nonideality coefficients from self-association properties, and thereby extends the quantitative interpretation of nonideality coefficients to probe dynamics in highly concentrated protein solutions.

Measuring aggregates, self-association, and weak interactions in concentrated therapeutic antibody solutions.

Monoclonal antibodies are a class of biotherapeutics used for an increasing variety of disorders, including cancer, autoimmune, neurodegenerative, and viral diseases. Besides their antigen specificity, therapeutic use also mandates control of their solution interactions and colloidal properties in order to achieve a stable, efficacious, non-immunogenic, and low viscosity antibody solution at concentrations in the range of 50-150 mg/mL. This requires characterization of their reversible self-association, aggregation, and weak attractive and repulsive interactions governing macromolecular distance distributions in solution. Simultaneous measurement of these properties, however, has been hampered by solution nonideality. Based on a recently introduced sedimentation velocity method for measuring macromolecular size distributions in a mean-field approximation for hydrodynamic interactions, we demonstrate simultaneous measurement of polydispersity and weak and strong solution interactions in a panel of antibodies with concentrations up to 45 mg/mL. By allowing approximately an order of magnitude higher concentrations than previously possible in sedimentation velocity size distribution analysis, this approach can substantially improve efficiency and sensitivity for characterizing polydispersity and interactions of therapeutic antibodies at or close to formulation conditions.

Nucleic acid-induced dimerization of HIV-1 Gag protein.

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

All tubulins are not alike: Heterodimer dissociation differs among different biological sources.

Tubulin, the subunit of microtubules, is a noncovalent heterodimer composed of one α- and one ß-tubulin monomer. Both tubulins are encoded by multiple genes or composed of different isotypes, which are differentially expressed in different tissues and in development. Tubulin αß dimers are found throughout the eukaryotes and, although very similar, are known to differ among organisms. We seek to investigate tubulins from different tissues and different organisms for a basic physical characteristic: heterodimer stability and monomer exchange between heterodimers. We previously showed that mammalian brain tubulin heterodimers reversibly dissociate, following the mass action law. Dissociation yields native monomers that can exchange with added tubulin to form new heterodimers. Here, we compared the dissociation of tubulins from multiple sources, including mammalian (rat) brain, cultured human cells (HeLa cells), chicken brain, chicken erythrocytes, and the protozoan Leishmania We used fluorescence-detected analytical ultracentrifugation to measure tubulin dissociation over a >1000-fold range in concentration and found that tubulin heterodimers from different biological sources differ in Kd by as much as 150-fold under the same conditions. Furthermore, when fluorescent tracer tubulins from various sources were titrated with unlabeled tubulin from a single source (rat brain tubulin), heterologous dimerization occurred, exhibiting similar affinities, in some cases binding even more strongly than with autologous tubulin. These results provide additional insight into the regulation of heterodimer formation of tubulin from different biological sources, revealing that monomer exchange appears to contribute to the sorting of α- and ß-tubulin monomers that associate following tubulin folding.

Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces.

The centerpiece of the sample cell assembly in analytical ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data.

A Reappraisal of Sedimentation Nonideality Coefficients for the Analysis of Weak Interactions of Therapeutic Proteins.

The study of weak or colloidal interactions of therapeutic proteins in different formulations allows prediction and optimization of protein stability. Various biophysical techniques have been applied to determine the second osmotic virial coefficient B2 as it reflects on the macromolecular distance distribution that governs solution behavior at high concentration. In the present work, we exploit a direct link predicted by hydrodynamic theory between B2 and the nonideality of sedimentation, commonly measured in sedimentation velocity analytical ultracentrifugation through the nonideality coefficient of sedimentation, kS. Using sedimentation equilibrium analytical ultracentrifugation for independent measurement of B2, we have examined the dependence of kS on B2 for model proteins in different buffers. The data exhibit the expected linear relationship and highlight the impact of protein shape on the magnitude of the nonideality coefficient kS. Recently, measurements of kS have been considerably simplified allowing higher throughput and simultaneous polydispersity assessment at higher protein concentrations. Thus, sedimentation velocity may offer a useful approach to compare the impact of formulation conditions on weak interactions and simultaneously on higher-order structure of therapeutic proteins.

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity.

Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid-liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun. 2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x ). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer-dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately -9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity.

In concentrated macromolecular solutions, weak physical interactions control the solution behavior including particle size distribution, aggregation, liquid-liquid phase separation, or crystallization. This is central to many fields ranging from colloid chemistry to cell biology and pharmaceutical protein engineering. Unfortunately, it is very difficult to determine macromolecular assembly states and polydispersity at high concentrations in solution, since all motion is coupled through long-range hydrodynamic, electrostatic, steric, and other interactions, and scattering techniques report on the solution structure when average interparticle distances are comparable to macromolecular dimensions. Here we present a sedimentation velocity technique that, for the first time, can resolve macromolecular size distributions at high concentrations, by simultaneously accounting for average mutual hydrodynamic and thermodynamic interactions. It offers high resolution and sensitivity of protein solutions up to 50 mg/ml, extending studies of macromolecular solution state closer to the concentration range of therapeutic formulations, serum, or intracellular conditions.

Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.

Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein-protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein-antibody complexes with picomolar KD values. Using an EGFP-nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein-protein interactions in the micromolar KD value range. Sedimentation experiments typically take ∼3 h of preparation and 6-12 h of run time, followed by data analysis (typically taking 1-3 h).

Crystal Structure of Chicken γS-Crystallin Reveals Lattice Contacts with Implications for Function in the Lens and the Evolution of the ßγ-Crystallins.

Previous attempts to crystallize mammalian γS-crystallin were unsuccessful. Native L16 chicken γS crystallized avidly while the Q16 mutant did not. The X-ray structure for chicken γS at 2.3 Å resolution shows the canonical structure of the superfamily plus a well-ordered N arm aligned with a ß sheet of a neighboring N domain. L16 is also in a lattice contact, partially shielded from solvent. Unexpectedly, the major lattice contact matches a conserved interface (QR) in the multimeric ß-crystallins. QR shows little conservation of residue contacts, except for one between symmetry-related tyrosines, but molecular dipoles for the proteins with QR show striking similarities while other γ-crystallins differ. In γS, QR has few hydrophobic contacts and features a thin layer of tightly bound water. The free energy of QR is slightly repulsive and analytical ultracentrifugation confirms no dimerization in solution. The lattice contacts suggest how γ-crystallins allow close packing without aggregation in the crowded environment of the lens.

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield.

Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation-properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Förster resonance energy transfer with transport methods.

Comparative insight into surfactants mediated amyloidogenesis of lysozyme.

Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

Protonation favors aggregation of lysozyme with SDS.

Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (∼11). Negatively charged SDS was used to 'antagonize' positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0-1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.

Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R h) of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.