Equilibrium Analytical Ultracentrifugation Research Articles

Structural Information on Supramolecular Copper(II) β-Diketonate Complexes from Atomic Force Microscopy and Analytical Ultracentrifugation.

Supramolecular Cu(II) complexes were prepared from two trifunctional β-diketone ligands. The ligands (CH3Si(phacH)3 and CH3Si(phprH)3, represented by LH3) contain three aryl-β-diketone moieties joined by an organosilicon group. The complexes have the empirical formula Cu3L2, as expected for combinations of Cu2+ and L3-. Several metal-organic polyhedra (MOPs) [Cu3L2]n are possible (n = 1-10); a dodecahedron (Cu30L20; n = 10; estimated diameter of ca. 5 nm) should be the most stable because its internal bond angles would come closest to ideal values. Atomic force microscopy (AFM), performed on samples deposited from solution onto mica substrates, revealed a distribution of sample heights in the 0.5-3.0 nm range. The most commonly observed heights were 0.5-1.5 nm, corresponding to the smallest possible molecules (Cu3L2, i.e., n = 1). Some molecular cubes (Cu12L8; ca. 2.5 nm) or larger molecules or aggregates may be present as well. Equilibrium analytical ultracentrifugation (AUC) was also used to probe the compounds. A previously reported reference compound, the molecular square Cu4(m-pbhx)4 (M = 2241 g mol-1), behaved well in AUC experiments in four nonpolar organic solvents. AUC data for the new tris(β-diketonate) MOPs [Cu3L2]n in toluene and fluorobenzene did not agree well with the theoretical results for a single solute. The data were fit well by a two-solute model, but these results were not consistent in the two solvents used, and some run-to-run variability was noted even in the same solvent. Also, the calculated molecular weights differed significantly from those expected for [Cu3L2]n ([Cu3(CH3Si(phac)3)2]n, multiples of 1322 g mol-1; or [Cu3(CH3Si(phpr)3)2]n, multiples of 1490 g mol-1).

Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation.

Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80min.

Experimental Method to Distinguish between a Solution and a Suspension

AbstractDispersion of objects in a fluid phase can be classified as solutions (Gibbs free energy of mixing, ΔGmix < 0) or suspensions (ΔGmix > 0) depending on their thermodynamic stability. Small objects tend to form solutions, larger ones suspensions, e.g., molecules versus micrometer‐sized colloids. Proteins and nanomaterials fall between these two size regimes. The long‐standing issue of whether proteins and nanoparticles are dissolved or suspended remains an important research question. Here, a simple, versatile, and experimentally robust method, based on sedimentation equilibrium analytical ultracentrifugation (SE‐AUC), which can determine whether proteins, nanoparticles, or polymers form solutions or suspensions, is presented. SE‐AUC determines the osmotic pressure profile for a dispersion. Such a profile for solutions (equilibrium one‐phase systems) is independent of the initial and the operating conditions. The opposite is true for suspensions that are nonequilibrium two‐phase systems. This study proves that bovine serum albumin and lysozyme form solutions while ferritin and apoferritin form suspensions.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 μM. As the KD of CBMCipA binding to cellulose is below 1 μM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.

Polysaccharide valproates: Structure - property relationships in solution

Polysaccharides are promising macromolecular platforms for use in the life sciences. Here, bioactive cellulose, pullulan, and dextran valproates are characterized hydrodynamically by sedimentation velocity and thermodynamically by sedimentation equilibrium analytical ultracentrifugation. Using sedimentation-diffusion analysis of sedimentation velocity experiments by numerical solution of the Lamm equation enabled the calculation of sedimentation and diffusion coefficients, and consequently molar masses. Sedimentation equilibrium experiments were then also used to determine the average molar masses. The corresponding set of data, with independently performed self-diffusion measurements by nuclear magnetic resonance spectroscopy, and together with size exclusion chromatography molar masses by coupling to refractive index-, viscometric-, and multi-angle laser light scattering detection, were subsequently correlated to each other by the hydrodynamic invariant and sedimentation parameter. We assess statistically most relevant average values of the molar masses of these polysaccharide valproates with relevant macromolecular conformational characteristics.

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.

Accounting for thermodynamic non-ideality in the Guinier region of small-angle scattering data of proteins.

Hydrodynamic studies of the solution properties of proteins and other biological macromolecules are often hard to interpret when the sample is present at a reasonably concentrated solution. The reason for this is that solutions exhibit deviations from ideal behaviour which is manifested as thermodynamic non-ideality. The range of concentrations at which this behaviour typically is exhibited is as low as 1–2 mg/ml, well within the range of concentrations used for their analysis by techniques such as small-angle scattering. Here we discuss thermodynamic non-ideality used previously used in the context of light scattering and sedimentation equilibrium analytical ultracentrifugation and apply it to the Guinier region of small-angle scattering data. The results show that there is a complementarity between the radially averaged structure factor derived from small-angle X-ray scattering/small-angle neutron scattering studies and the second virial coefficient derived from sedimentation equilibrium analytical ultracentrifugation experiments.

Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) is a gold standard for the rigorous determination of macromolecular buoyant molar masses and the thermodynamic study of reversible interactions in solution. A significant experimental drawback is the long time required to attain SE, which is usually on the order of days. We have developed a method for time-optimized SE (toSE) with defined time-varying centrifugal fields that allow SE to be attained in a significantly (up to 10-fold) shorter time than is usually required. To achieve this, numerical Lamm equation solutions for sedimentation in time-varying fields are computed based on initial estimates of macromolecular transport properties. A parameterized rotor-speed schedule is optimized with the goal of achieving a minimal time to equilibrium while limiting transient sample preconcentration at the base of the solution column. The resulting rotor-speed schedule may include multiple over- and underspeeding phases, balancing the formation of gradients from strong sedimentation fluxes with periods of high diffusional transport. The computation is carried out in a new software program called TOSE, which also facilitates convenient experimental implementation. Further, we extend AUC data analysis to sedimentation processes in such time-varying centrifugal fields. Due to the initially high centrifugal fields in toSE and the resulting strong migration, it is possible to extract sedimentation coefficient distributions from the early data. This can provide better estimates of the size of macromolecular complexes and report on sample homogeneity early on, which may be used to further refine the prediction of the rotor-speed schedule. In this manner, the toSE experiment can be adapted in real time to the system under study, maximizing both the information content and the time efficiency of SE experiments.

Sedimentation in a Time-Varying Centrifugal Field for Rapid Attainment of Sedimentation Equilibrium

Sedimentation equilibrium (SE) analytical ultracentrifugation is a gold standard for the rigorous thermodynamic study of buoyant molecular weight and reversible interactions of macromolecules in solution. A significant drawback is the long experiment time, as it takes days to attain SE with standard solution columns. We have developed a new method for using a time-varying centrifugal field optimized such as to attain SE in significantly shorter time than usually required. Experimental data show that this permits long-column SE experiments to be carried out in times comparable to sedimentation velocity experiments, approximately fivefold shorter than standard SE. In contrast to the classical initial overspeeding method, which uses a single initial speed, we employ a freely varying rotor speed profile during an initial phase, for example, parameterized as a step-wise modulated exponential decay to the desired SE rotor speed. The rotor speed schedule is computationally optimized on the basis of numerical Lamm equation solutions for given macromolecular sedimentation parameter estimates, with the goal to provide a rapid attainment of equilibrium without the drawback of strong transient sample pre-concentration at the base of the solution column. The resulting rotor speed schedule frequently includes both over- and under-speeding sequences, and can be conveniently implemented on the Optima XLA/I analytical ultracentrifuge. We extended AUC data analysis models in SEDFIT to permit the analysis of concentration profiles in arbitrarily time-varying fields, to make it possible to exploit the migration in the initially high centrifugal field for estimates on macromolecular sedimentation parameters, which may be used in real-time to refine the prediction of the rotor speed schedule, so that the SE experiment can be optimized in both information content and time efficiency.

Characterization of Homogeneous, Cooperative Protein-DNA Clusters by Sedimentation Equilibrium Analytical Ultracentrifugation and Atomic Force Microscopy.

Strong, positively cooperative binding can lead to the clustering of proteins on DNA. Here, we describe one approach to the analysis of such clusters. Our example is based on recent studies of the interactions of O(6)-alkylguanine DNA alkyltransferase (AGT) with high-molecular-weight DNAs (Adams et al., 2009; Tessmer, Melikishvili, & Fried, 2012). Cooperative cluster size distributions are predicted using the simplest homogeneous binding and cooperativity (HBC) model, together with data obtained by sedimentation equilibrium analysis. These predictions are tested using atomic force microscopy imaging; for AGT, measured cluster sizes are found to be significantly smaller than those predicted by the HBC model. A mechanism that may account for cluster size limitation is briefly discussed.

Characterization of Capsular Polysaccharides and Their Glycoconjugates by Hydrodynamic Methods.

Hydrodynamic methods are relevant for the characterization of carbohydrates such as capsular bacterial polysaccharides or glycoconjugates in solution. This chapter focuses on the following hydrodynamic methods: sedimentation velocity analytical ultracentrifugation (SV AUC), dynamic light scattering (DLS), sedimentation equilibrium analytical ultracentrifugation (SE AUC), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and capillary viscometry-intrinsic viscosity measurement. The chapter highlights the general principle of these five methods, describes experimental details, and specifies advances in the last years.

SEDFIT-MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge.

Sedimentation equilibrium (analytical ultracentrifugation) is one of the most inherently suitable methods for the determination of average molecular weights and molecular weight distributions of polymers, because of its absolute basis (no conformation assumptions) and inherent fractionation ability (without the need for columns or membranes and associated assumptions over inertness). With modern instrumentation it is also possible to run up to 21 samples simultaneously in a single run. Its application has been severely hampered because of difficulties in terms of baseline determination (incorporating estimation of the concentration at the air/solution meniscus) and complexity of the analysis procedures. We describe a new method for baseline determination based on a smart-smoothing principle and built into the highly popular platform SEDFIT for the analysis of the sedimentation behavior of natural and synthetic polymer materials. The SEDFIT-MSTAR procedure - which takes only a few minutes to perform - is tested with four synthetic data sets (including a significantly non-ideal system), a naturally occurring protein (human IgG1) and two naturally occurring carbohydrate polymers (pullulan and λ-carrageenan) in terms of (i) weight average molecular weight for the whole distribution of species in the sample (ii) the variation in "point" average molecular weight with local concentration in the ultracentrifuge cell and (iii) molecular weight distribution.

Allosteric Regulation of the Epidermal Growth Factor Receptor

It is well established that aberrant activation of the Epidermal Growth Factor Receptor (EGFR) drives cellular transformation and oncogenesis. A series of activating mutations in the extracellular region of EGFR have been identified in glioblastoma multiforme (GBM) patients, and shown to transform NIH 3T3 cells. Sedimentation equilibrium analytical ultracentrifugation analysis demonstrates that, in the context of the soluble EGFR extracellular region (sEGFR), these mutations are monomeric in the absence of ligand and dimerize upon ligand binding with affinities that are similar to those observed for the wildtype receptor. We also find that these mutations elevate the affinity of growth factor binding to sEGFR, and that there are differential effects on the entropic driving forces of growth factor binding to sEGFR. Taken together, these results demonstrate a potentially novel mode of EGFR disregulation by these GBM mutations. Our recent detailed thermodynamic analysis of ligand binding and dimerization in the EGFR system suggests that a greater understanding of the changes that occur upon binding of ligand to EGFR will be required before we can appreciate the mechanisms of disregulation by oncogenic mutation. We will present our progress towards determining a profile of the dynamic and conformational effects of ligand binding to EGFR using associated hydrogen/deuterium exchange coupled to mass spectrometry (HX/MS). Comparing these profiles to those for EGFR bearing oncogenic mutations may reveal why these single amino acid substitutions can lead to receptor activation. Progress towards the application of these techniques to near full length receptors reconstituted in a lipid nanodisc model membrane system will also be reported, which will provide valuable information about the structurally poorly characterized juxtamembrane regions of EGFR.

Effect of Pegylation on Self-Association of IFN-α2b

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

The Oligomeric State of the Active Vps4 AAA ATPase

The cellular ESCRT (endosomal sorting complexes required for transport) pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including the human immunodeficiency virus. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type Saccharomyces cerevisiae and Sulfolobus solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size-exclusion chromatography and equilibrium analytical ultracentrifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p–Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double-ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.

Computational de Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4'-ethynyl-(2,2';6',2″-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2';6',2″-terpyridine)(2+) (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β1340 = 3100 × 10(-30) esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality.

Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation

AbstractModern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size‐distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self‐ and hetero‐association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective‐particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent‐solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1‐20.12.49. © 2013 by John Wiley & Sons, Inc.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Equilibrium Binding of Wild-type and Mutant Drosophila Heat Shock Factor DNA Binding Domain with HSE DNA Studied by Analytical Ultracentrifugation

We have investigated binding between wild-type and mutant Heat Shock Factor (HSF) DNA binding domains (DBDs) with 17-bp HSE containing a central 5'-NGAAN-3' element by equilibrium analytical ultracentrifugation using multi-wavelength technique. Our results indicate that R102 plays critical role in HSE recognition and the interactions are characterized by substantial negative changes of enthalpy () and entropy () with free energy change, of . N105 plays minor role in the HSE interactions with of , of and of , which are similar to those observed for wild-type DBD:HSE interactions (, and ) indicating higher entropy contribution for both wild-type and N105A DBD bindings to the HSE.

Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle

Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV requires fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein, GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔG(unf,H(2)O), of 33.4 ± 2.5 kcal/mol and melting temperature, T(m), of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH, with higher stability under lower-pH conditions (T(m) values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. On the basis of these results, we hypothesize that the pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism for controlling conformational preferences such that the six-helix bundle "postfusion" state is preferred under conditions of appropriately matured endosomes.