Monomer-dimer Self-association Research Articles

Energetic Dissection of Mab-Specific Reversible Self-Association Reveals Unique Thermodynamic Signatures.

Reversible self-association (RSA) remains a challenge in the development of therapeutic monoclonal antibodies (mAbs). We recently analyzed the energetics of RSA for five IgG mAbs (designated as A-E) under matched conditions and using orthogonal methods. Here we examine the thermodynamics of RSA for two of the mAbs that showed the strongest evidence of RSA (mAbs C and E) to identify underlying mechanisms. Concentration-dependent dynamic light scattering and sedimentation velocity (SV) studies were carried out for each mAb over a range of temperatures. Because self-association was weak, the SV data were globally analyzed via direct boundary fitting to identify best-fit models, accurately determine interaction energetics, and account for the confounding effects of thermodynamic and hydrodynamic nonideality. mAb C undergoes isodesmic self-association at all temperatures examined, with the energetics indicative of an enthalpically-driven reaction offset by a significant entropic penalty. By contrast, mAb E undergoes monomer-dimer self-association, with the reaction being entropically-driven and comprised of only a small enthalpic contribution. Classical interpretations implicate van der Waals interactions and H-bond formation for mAb C RSA, and electrostatic interactions for mAb E. However, noting that RSA is likely coupled to additional equilibria, we also discuss the limitations of such interpretations.

Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.

While humans lack the biosynthetic pathways for meso-diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum (VsDapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso-diaminopimelate. Kinetic studies show that VsDapL follows a Michaelis-Menten mechanism, with a KMapp of 4.0 mM toward its substrate l,l-diaminopimelate. The kcat (0.46 s-1) and the kcat/KM (115 s-1 M-1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that VsDapL exists in a monomer-dimer self-association, with a KD2-1 of 7.4 μM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in VsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

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.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

Evidence for a monomer-dimer equilibrium in native human Factor H

Factor H (FH) plays a central role in the regulation of complement activation via the alternative pathway. It is composed of 20 short complement regulator (SCR) domains. Previously, we had demonstrated that Factor H is dimeric in solution by X-ray and neutron scattering (Perkins et al. (1991) Biochemistry, 30, 2847-2857), while the follow-up study demonstrated that Factor H is monomeric (Aslam & Perkins (2001) J. Mol. Biol. 309, 1117-1138). Recently the SCR-6/8 and SCR-16/20 fragments of Factor H have each revealed monomer-dimer self-association with KD values of 40 μM and 16 μM respectively. We have reinvestigated the discrepancy in the earlier studies by the use of c(s) size-distribution analyses of Factor H in sedimentation velocity experiments on the analytical ultracentrifuge. Between 0.1 mg/ml to 0.6 mg/ml of Factor H in either phosphate buffer saline or Hepes buffer, two peaks were observed in the c(s) analyses between 25k r.p.m. and 50k r.p.m. rotor speeds. The major peak at 5.7 ± 0.1 S corresponds to a molecular weight at 142 ± 2 kDa, while the second peak at 9.2 ± 0.5 S corresponds to a molecular weight at 285 ± 24 kDa. The relative intensity of the second peak increased with concentration. We conclude that Factor H exists in a monomer-dimer equilibrium in solution. A tentative explanation of the outcomes of the 1991 and 2001 studies is that these were fortuitously performed in concentration ranges that corresponded to predominantly dimeric and monomeric Factor H. Further data collection is in progress and will be presented alongside a reanalysis of our 2001 data. From the results so far obtained, we conclude that Factor H will be predominantly monomeric in plasma in its physiological concentration range between 0.2 mg/ml to 0.8 mg/ml.

A Consistent Determination of the Dimerization Constants of the Self-Association of 2,2-Dimethyl-3-ethyl-3-pentanol in Carbon Tetrachloride from its Infrared Spectral Data

Two equations of linear type (Eqs. 10 and 17 in the text) have been derived to analyze the IR data to determine the dimerization constant consistently. Equation 10 is to be used to fit the integrated absorbances of the monomer band to obtain the molar monomer absorptivity, ϵm, and dimerization constant, K; Eq. 17 is to be used to fit the integrated absorbances of the dimer bands to obtain the molar dimer absorptivity, ϵd, and dimerization constant, K. Thus the same dimerization constant can be independently determined either from the monomer band or from the dimer band. The discrepancy between the two determined values provides an assessment of the consistency of determination. The monomer-dimer self-association of 2,2-dimethyl-3-ethyl-3-pentanol in the solvent of carbon tetrachloride was chosen to demonstrate the utility of these two equations.

Behavior of Tn3 Resolvase in Solution and Its Interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K D 1 − 2 = 50 μ M . Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of γδ resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.

New Method to Analyse IR-Studies of Monomer-Dimer Self-Association: 2,2-Dimethyl-3-Ethyl-3-Pentanol in Carbon Disulfide as an Example

Abstract Two new equations have been derived to treat the IR data from the monomer-dimer self-association. Equation (9), in the text, is to regress the integrated absorbances of the monomer bands to determine the molar absorptivity of the monomer band, ε m, and the dimerization constant, K. Equation (13), in the text, is to regress those of dimer band to obtain the molar absorptivity of the dimer band, ε d, and dimerization constant K. The self-association in the dilute solution of 2,2-dimethyl-3-ethyl-3-pentanol in carbon disulfide at different temperatures has been used as an example to demonstrate the usage of these two equations. The discrepancy between the values of K at the same temperature determined either from the monomer band or from the dimer band can be used as a criterion for the goodness of determination. The standard enthalpy and entropy of dimerization have also been obtained via a van’t Hoff plot from the data of K at different temperatures. The advantages of this new approach have also been discussed.

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)

The temperature-dependent self-association of β-lactoglobulin C in glycine buffers

The self-association of β-lactoglobulin C at low pH (ca. 2.5) in glycine buffers has been studied at four temperatures, 10, 16, 20, and 25 °C, by low- and high-speed sedimentation equilibrium experiments. One buffer had an ionic strength of 0.1 and the other an ionic strength of 0.2. With either buffer the concentration dependence of the apparent weight average molecular weight, M wa, was characteristic of a nonideal self-association. Like its genetic variants, β-lactoglobulin A and B, the self-association of β-lactoglobulin C increased with decreasing temperature; however, at the same temperature the association was always stronger in the buffer having the higher ionic strength. Several models were used to test the self-association, and a monomer-dimer self-association seemed to describe the self-association best with either buffer. Values of the association equilibrium constant, K 2, and the second virial coefficient, BM 1, are reported at each temperature for both series of experiments. Values of the thermodynamic functions, ΔG °, ΔH °, and ΔS °, are also reported for these experiments.