# Enhancing biological SAXS by solvent contrast variation: effect on structural stability and enzymatic activity

Viviam da Silva, Aurélien Thureau, Dominique Madern, Bruno Franzetti, Wanius Garcia, Javier Pérez, Eric Girard, Frank Gabel

► To cite this version:

Viviam da Silva, Aurélien Thureau, Dominique Madern, Bruno Franzetti, Wanius Garcia, et al. Enhancing biological SAXS by solvent contrast variation: effect on structural stability and enzymatic activity. Biophysical Journal, 2025, 10.1016/j.bpj.2025.11.009. hal-05377907

HAL Id: hal-05377907

https://hal.science/hal-05377907v1

Submitted on 22 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Enhancing biological SAXS by solvent contrast variation:
effect on structural stability and enzymatic activity

Viviam M. Da Silva¹, Aurélien Thureau², Dominique Madern¹, Bruno Franzetti¹, Wanius Garcia³, Javier Pérez², Eric Girard¹, Frank Gabel¹,⁴*

¹IBS, CEA, CNRS, UGA, 71 Avenue des Martyrs, 38000 Grenoble, France

²Synchrotron SOLEIL, L'Orme des merisiers, Départementale 128, 91190 Saint Aubin,
91192 Gif-sur-Yvette, France

³Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP,
Brazil

⁴ILL, 71 Avenue des Martyrs, 38000 Grenoble, France

*Correspondence: frank.gabel@ibs.fr, gabelfrank@ill.fr

Condensed running title:

Solvent contrast variation in BioSAXS
Abstract

Small-angle X-ray scattering (SAXS) has evolved over the past decades into an essential structural biology technique for the study of conformational states of solubilized biomacromolecules and assemblies. It complements a multitude of other methods, including the atomic-resolution techniques NMR, crystallography and Cryo-EM. In the vast majority of applications, the biological systems of interest are measured by SAXS in aqueous buffers. This approach, while quick and straightforward, limits the information content of the internal topology of biomacromolecular assemblies of variable electron density, such as protein-RNA/DNA complexes, and detergent/lipid-solubilized membrane proteins.

These shortcomings can be partially overcome by altering the solvent electron density by the addition of small, electron-rich molecules such as sugar, glycerol, salt and heavy-atom compounds, so-called contrast agents. A crucial issue in these contrast-variation SAXS experiments is the question whether the conformational state and the enzymatic activity of biomolecules are maintained after long incubation times with contrast agents, which are often required at molar concentrations.

Here we present a structural SAXS study of two proteins, a bacterial endo-β-1,4-mannanase and a malate dehydrogenase, in the presence of high concentrations of sucrose and a lanthanide compound, and evaluate the residual enzymatic activity as a function of the incubation time. Our results indicate that the global conformation and the oligomeric state of both proteins is preserved over the entire concentration range of both contrast agents, and that their enzymatic activities are unaltered after long incubation times (up to 24 hours). Taken together, our results encourage a more routine use, as well as further developments, of contrast variation SAXS experiments for structural studies of a variety of important and challenging biomacromolecular assemblies.

Statement of significance

SAXS is an essential structural biology technique, in complement to NMR, crystallography, Cryo-EM and others. While quick and straightforward, the usual aqueous buffer conditions limit the information content on the internal topology of biomacromolecular assemblies such as protein-RNA/DNA complexes, and detergent/lipid-solubilized membrane proteins.

These shortcomings can be partially overcome by varying the solvent electron density by the addition of small, electron-rich molecules (so-called contrast agents). A crucial issue in such contrast-variation SAXS experiments is whether the conformational state and the enzymatic activity of biomolecules are impacted.

Here we present a SAXS study on two enzymes in the presence of two different contrast agents. Our results encourage the further use and future developments of SAXS contrast variation in structural biology.
Introduction

Biological small-angle X-ray scattering (BioSAXS) has come to maturity over the past
decades and represents an indispensable contribution to the structural biology toolbox for the
study of biomacromolecules and supramolecular assemblies in solution (1, 2). In recent years,
new approaches in electron microscopy and tomography, as well as atomic structure
predictions, have revolutionized structural biology (3, 4). Nevertheless, BioSAXS remains a
valuable and powerful tool to probe hypotheses on macromolecular conformations, flexibility,
and interactions under near-physiological conditions in solution (5), as a function of solvent
properties (pH, salinity...) and other physico-chemical parameters, including temperature (6).
While BioSAXS data is now being recorded routinely at synchrotrons and home sources, and
the final 1D scattering curves can be reduced efficiently and swiftly with the help of data
reduction pipelines (7), most scattering data of biological systems are usually limited to
concentration series of the biomolecule(s) of interest in aqueous buffer conditions. This
specific experimental condition, while sufficient in many cases, represents certain limits to the
data analysis, in particular of complex biomacromolecular assemblies composed of multiple
partners of different electron densities, e.g. protein-RNA/DNA or lipid/detergent-solubilized
membrane protein complexes (8). The difficulty consists in correctly extracting the
contribution of each partner in order to attribute the correct conformation and the respective
location and orientation of each partner within the assembled complex, which increases the
ambiguity of structural models that are in agreement with the experimental data (9).

Small-angle neutron scattering (SANS), in combination with H₂O/D₂O solvent contrast variation and labeling (i.e. deuteration) of molecules, can address this issue by obtaining specific structural information from individual partners within an assembled complex, while masking the signals from others (so-called “contrast-matching”) (10). Neutrons are mostly scattered by the atomic nuclei and the scattering process is sensitive on the isotope, in particular on H-D exchange. Importantly, solvent contrast variation in SANS can be tuned to match the neutron scattering length density of any biomolecule of interest, in particular proteins, RNA, DNA, and lipids. Furthermore, deuteration can be used to modulate contrast of biomolecules such as proteins, RNA/DNA and lipids, and to distinguish molecules of similar chemical composition, e.g. two proteins (11). The impact of D₂O and deuteration on the structural integrity of biomolecules has been studied by neutron scattering, and SANS in particular: while H₂O/D₂O solvent exchange is generally considered a relatively “gentle” approach, high percentages of D₂O may lead to an alteration of enzymatic activity and/or solubility of proteins (12).

In SAXS, contrast variation (CV) is more challenging to implement. X-rays are sensitive to the electron density differences between solubilized molecules and the solvent. The solvent electron density can be modified by the addition of small, electron-rich molecules such as glycerol (13), sugar (6, 14, 15) and salt (16–18) (so-called “contrast agents”) with respect to the electron density of solubilized biomolecules (6, 19, 20).

CV-SAXS experiments using “classical” contrast agents (sugar, glycerol, salt...) are,
however, limited by the solvent electron densities achievable, which allow to equal those of
proteins, but do, in general, not reach those of RNA/DNA, in contrast to SANS. As an
example, protein electron densities of 0.41-0.42 e/Å³ are reached in 2 M sugar solutions (21,
22). Luzzati et al. reported CV-SAXS experiments on low density serum lipoprotein (LDL),
where the authors were able to exceed that limit by working in 6 M NaBr, reaching a solvent
electron density of 0.443 e/Å³ (16). Salt solutions at molar concentrations, however, may
have deleterious impacts on the structural integrity of some biomolecules (18).

Interestingly, new classes of more efficient molecules have been probed recently as
alternative SAXS contrast agents. Lanthanide-based complexes, in particular, have yielded
promising results on proteins, detergent micelles and a protein-RNA complex, reaching solvent electron densities of 0.43 e/Å³ (23). A complex based on iodine yielded even higher solvent electron densities (0.455 e/Å³), but disrupted the structural integrity of proteins and detergent micelles (24). These recent developments show, however, that CV-SAXS has the capacity to reach electron densities between those of proteins and RNA/DNA, and thus represents an attractive development to complement SANS, as well as standard SAXS experiments in aqueous buffers. CV-SAXS therefore has a huge potential for applications in biology, considering the high throughput and good signal-to-noise ratio, the dense network of existing synchrotron beamlines and table-top SAXS instruments, and the contrast conditions in SAXS, which are complementary to those in SANS experiments.

An important issue to be addressed before a more routine use of these new classes of molecules, as well as classical SAXS contrast agents, is their compatibility with the structural integrity and biological function of biomacromolecules and assemblies of interest. This point is also of practical interest since contrast agents are usually necessary at molar concentrations in order to match the electron densities of proteins, and a structural stability of biomacromolecules is required over minutes up to hours during typical SAXS experiments.

In the present study we address these important questions by studying the structural and functional properties of two enzymes, the bacterial endo-β-1,4-mannanase (EC 3.2.1.78) from *Thermotoga petrophila* (TpMan), and the malate dehydrogenase (EC.1.1.1.37) of *Chloroflexus aurantiacus* (CaMalDH). SAXS data are recorded for both enzymes in the presence of various concentrations of two contrast agents, sucrose and GdHPDO3A, up to molar concentrations. In addition, residual activity measurements are conducted for both proteins after long (up to 24 hours) incubation times to both contrast agents, and correlated with the structural information obtained by the SAXS data.

Taken together, our data suggest that both enzymes maintain their structural integrity (i.e. conformation and oligomeric state) in the presence of both contrast agents over the whole concentration ranges tested, and that their functional capacity is not alleviated after long incubation times. Our results are very encouraging for a more extensive use and further developments of CV-SAXS experiments for the structural study of a broad variety of challenging and important biomacromolecular assemblies.

# Material and Methods

## EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS

### Truncated endo-β-1,4-mannanase from *Thermotoga petrophila* (Tp-GH5-Ig):

The bacterial endo-β-1,4-mannanase (EC 3.2.1.78) from *Thermotoga petrophila* (TpMan) is an enzyme composed of 667 amino acid residues (Gene Bank: ABQ47550.1) with a molecular weight of 73 kDa. It consists of an N-terminal GH5 catalytic domain (belonging to the subfamily 7) and two ancillary domains. The central ancillary domain is a thermostabilizing immunoglobulin-like module (Ig) of unknown function and the C-terminal is a carbohydrate-binding module (CBM) belonging to the CBM27 family (25).

The expression and purification of a monomeric truncated construct of 53 kDa, where the CBM27 was removed ("Tp-GH5-Ig", amino acid residues 32 to 495), were carried out as described previously with minor modifications (25). Briefly, 1 L of Luria-Bertani (LB) broth medium containing kanamycin (50 µg/mL), inoculated with *Escherichia coli* BL21(DE3) and containing the expression vector, was incubated at 37 °C until reaching an optical density (OD) of 0.5 at 600 nm. Subsequently, the culture was induced with 1 mM Isopropyl β-D-1-
thiogalactopyranoside (IPTG) for 4 h at 37 °C. Next, the culture was harvested by centrifugation (5,000×g for 20 min at 4 °C), resuspended in 20 mM Tris-HCl buffer adjusted at pH 7.4, and ruptured by sonication. The suspension was then centrifuged at 5,000×g for 20 min at 4 °C to separate the cell debris. The supernatant was heat-treated (at 70 °C for 30 min) to precipitate heat-labile proteins and centrifuged again at 5,000×g for 20 min and 4 °C.

After the heat-shock, Tp-GH5-Ig was purified by affinity chromatography using the Ni-NTA column equilibrated with 20 mM Tris-HCl buffer adjusted at pH 7.4. The recombinant Tp-GH5-Ig was then eluted from the column with the same buffer containing 150 mM imidazole. The His6-tag was removed by overnight digestion with bovine Thrombin protease (CAS 9002044 - Sigma-Aldrich). Finally, the Tp-GH5-Ig without the His6-tag was purified by gel filtration through prep-grade Superdex 200 column (GE-healthcare) and the elution was carried out with 50 mM Tris-HCl and 150 mM NaCl buffer adjusted at pH 7.4. Active fractions detected by using the standard enzymatic assay were pooled, and then concentrated to 17 mg/mL using an Amicon PM30 filter and stored at 4 °C.

## Malate dehydrogenase from *Chloroflexus aurantiacus* (CaMalDH):

The malate dehydrogenase (EC 1.1.1.37) from *Chloroflexus aurantiacus* (CaMalDH) is a tetrameric enzyme composed of 309 amino acid residues per subunit with a molecular weight of 36 kDa per monomer. It contains a NAD(P)-binding Rossmann-fold domain (residues 1 to 138) and a L-lactate C-terminal-like domain (residues 146 to 308) (26).

E. coli BL21 (DE3)pLysS competent cells were transformed with the pET-20 expression vector encoding the *C. aurantiacus* MalDH gene protein. Mutants were selected by growth on LB agar plates containing 100 µg/mL ampicillin. For overexpression of CaMalDH, a single colony was grown overnight at 37 °C in 50 mL LB medium containing 100 µg/mL ampicillin. Twenty milliliters of this culture were used to inoculate 2 L of LB medium containing 100 µg/mL ampicillin. The cells were grown at 37 °C until the OD at 600 nm reached 0.6. IPTG was added to a final concentration of 0.5 mM to induce protein expression, and the culture was incubated for an additional 4 h at 37 °C. Bacterial cells were harvested by centrifugation at 6,000×g for 20 min at 4 °C. The pellet was resuspended in 60 mL of 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl. The cells were lysed by sonication and subsequently centrifuged at 6,000×g for 20 min. The supernatant was incubated at 70 °C for 30 min, followed by another centrifugation at 6,000×g for 20 min.

The soluble fraction was loaded onto a Q Sepharose column equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl. The protein was eluted using a linear gradient of 0-1 M NaCl. Fractions containing CaMalDH were concentrated and injected onto a Superdex 200 gel filtration column equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl. The fractions containing the protein were pooled and loaded onto a hydrophobic interaction chromatography column (Phenyl HP) equilibrated in 2 M ammonium sulfate containing 50 mM Tris-HCl (pH 7.4). The protein was eluted using a linear gradient from 2-0 M ammonium sulfate, continuing until the buffer was 100% 50 mM Tris-HCl (pH 7.4) with 50 mM NaCl. Active fractions detected using the standard enzymatic assay (see next section) were pooled and dialyzed against 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl, then concentrated to 12.3 mg/mL using an Amicon PM50 filter and stored at 4 °C. After purification, the purities of both Tp-GH5-Ig and CaMalDH were verified by 12% SDS-PAGE. The concentrations of the proteins were determined by absorbance at 280 nm using theoretical extinction coefficients based on the amino acid composition (27). The theoretical coefficients employed were 113,220 M⁻¹ cm⁻¹ for Tp-GH5-Ig and 189,100 M⁻¹ cm⁻¹ for CaMalDH.
## STANDARD ENZYMATIC ASSAYS

For mannanase activity measurement, 3 µL of the enzyme mixture was used at each time point and concentration. This was mixed with 15 µL of 1% locust bean galactomannan (CAS 9000-40-2, Sigma-Aldrich) as the substrate and 12 µL of 100 mM Tris-HCl buffer (pH 7.4), resulting in a final reaction volume of 30 µL. The reaction mixture, representing the degradation of the polysaccharide, was incubated at 90 °C for 10 min, and the reaction was then stopped by adding dinitrosalicylic acid (DNS) reagent (28). The DNS reacts with the reducing sugars released during the enzymatic degradation, resulting in the formation of a reddish-brown complex. The samples were subsequently boiled at 100 °C for 10 min to enhance the development of the colored complex, which was then measured at 540 nm. Afterward, the samples were diluted 3-fold with water, and their absorbance at 540 nm was measured using a BioTek Synergy H4 microplate reader (BioTek Instruments, Winooski, VT, USA). All experiments were performed in quadruplicate, and the average values are reported. For malate dehydrogenase activity measurement, 5 µL of the enzyme mixture was used at each time point and concentration. This was mixed with 20 µL of NADH (0.5 mM) and 20 µL of oxaloacetate (OAA, CAS 015789.04, Sigma-Aldrich). The reaction was carried out at 60 °C in a final volume of 500 µL, using a 10 mM Tris, 50 mM NaCl buffer at pH 7.2 to achieve the desired volume. In this reaction, OAA acts as the substrate, which is reduced to malate, while NADH serves as the electron donor and is oxidized to NAD+. The reaction was monitored at 340 nm by tracking the oxidation of NADH, as it strongly absorbs at this wavelength. The rate of change in absorbance at 340 nm per min (ΔAbs 340 nm/min) was used to plot the results. All measurements were performed in triplicate.

## RESIDUAL ACTIVITY MEASUREMENTS

To monitor the effects of various incubation periods at different concentrations of contrast agents, we monitored the residual activity as described below.

The stock solutions of the contrast agents were: sucrose (catalog number S7903 Sigma-Aldrich) at 2.4 M, and GdHPDO3A (Gadoteridol) (catalog number 1287631 Sigma-Aldrich) at 1.2 M. They were prepared in 100 mM Tris-HCl buffer (pH 7.4) and in 10 mM Tris-HCl buffer (pH 7.2) containing 50 mM NaCl for the Tp-GH5-Ig and CaMalDH activity assays, respectively. The protein concentration stock was 3.5 mg/mL for Tp-GH5-Ig and 1.5 mg/mL for CaMalDH.

For mannanase activity, the incubation was conducted using a final Tp-GH5-Ig concentration of 0.70 mg/mL. First, the enzyme was exposed to the contrast agents sucrose and GdHPDO3A at concentrations of 100, 300, 500, 750, and 960 mM in several time points as 0, 3, 5 and 24 hours at room temperature. The reaction was carried out in a final volume of 50 µL, achieved by using 100 mM Tris-HCl buffer (pH 7.4). A condition with no contrast agent (0 mM) was included as a positive control. After the various incubation periods, an aliquot of 3 µL of the solution was tested using the standard mannanase enzymatic assay. All measurements were performed in quadruplicate, and the average values are reported.

For malate dehydrogenase activity, the incubation was performed using CaMalDH at a final concentration of 0.076 mg/mL. First, the enzyme was exposed to the contrast agent's sucrose and GdHPDO3A at final concentrations of 100, 300, 500, 750, and 960 mM in several time points as 0, 3, 5 and 24 hours at room temperature. The reaction was carried out in a final volume of 60 µL, achieved by using 10 mM Tris-HCl buffer (pH 7.2) containing 50 mM NaCl. A control condition with no contrast agent (0 mM) was included as a positive control. At the end of each incubation period, an aliquot of 5 µL of the solution was tested using the standard malate dehydrogenase enzymatic assay. The rate of change in absorbance at 340 nm
per minute (ΔAbs 340 nm/min) was used to plot the results. All measurements were
performed in triplicate.

The detailed protocol and amounts of materials mixed for all enzymatic assays and residual measurements are provided in Tables S1 to S4.

## SMALL ANGLE X-RAY SCATTERING

### Sample preparation

Sucrose and GdHPDO3A contrast agent stock solutions were prepared by weighing a specific amount of contrast agent on a Mettler Toledo Excellence high-precision balance and adding Milli-Q water for GdHPDO3A and protein buffer for sucrose by pipetting. The final volume of the mixture was determined by pipetting.

For each series of GdHPDO3A, solutions were sequentially lyophilized overnight with appropriate volume ratios to achieve the desired concentration, using an Eppendorf Concentrator Plus instrument. While sucrose can be lyophilized, the results are not optimal in terms of homogeneity, flakiness, and removal of liquid, so the stock solutions were directly diluted with buffer to prepare the desired concentrations. It should be noted that the use of lyophilization for GdHPDO3A is advantageous since it allows reaching higher final protein concentrations with respect to a dilution approach from a stock solution, as in the case of sucrose.

The lyophilized GdHPDO3A powder aliquots and sucrose stock solution aliquots were mixed with protein and/or buffer to reach a final volume of 50 µL. The detailed protocols and amounts of materials mixed are provided in Tables S5 and S6 for 1.189 M GdHPDO3A and Tables S7 and S8 for 2.407 M sucrose, which served as a reference solution for calculating and calibrating solvent electron densities.

### SAXS experiments, data reduction and analysis

All CV-SAXS experiments were carried out in batch mode using an optimized configuration on the SWING beamline at SOLEIL synchrotron (Saint Aubin, France) (29). In order to minimize absorption from the lanthanide complexes, the X-ray energy was set to 15.5 keV (λ=0.8 Å), and a thermalized (20 °C) quartz capillary with a diameter of 1.0 mm and a wall thickness of 10 µm was employed. This setup also minimizes capillary fouling, and reduces parasitic scattering at low q-values. A sample-to-detector distance of 2.00 m was used, covering a momentum transfer range of 0.004 < q [Å⁻¹] < 0.717, with q=4πsinθ/λ, where 2θ is the scattering angle. For each sample, a volume of 40 µL was injected at a flow rate of 0.84 µL/min using an automated robotic arm, developed recently on the SWING beamline (30). Data acquisition was performed at a rate of 1 frame per second, with an exposure time of 0.99 s.

The 2D scattering patterns were reduced and normalized using a water reference into 1D intensities using the Foxtrot software (31), typically 20–25 frames were binned, depending on the dataset, after manually checking for radiation damage and validation of identical transmissions. Buffer intensities were subtracted from sample intensities using PRIMUS (32). In the case of sucrose, this step was performed using manual buffer corrections, which varied depending on the specific case, due to slight sucrose concentration variation between samples containing the protein or not. For some samples, a manual constant buffer adjustment relative to the protein curve was required, typically of the order of 1%, while for others no correction was needed (Tables S9 and S10).

For the GdHPDO3A solutions, X-rays are strongly absorbed, and buffers were subtracted after careful calibration against the measured transmissions, following a previously established protocol (24). Briefly, the contrast-agent concentrations in both the sample and the
buffer solutions were calibrated against a reference curve, and the buffers were corrected to match the sample concentrations by using a linear interpolation between adjacent data points (Figures S1 to S4).

For all (sucrose and GdHPDO3A) samples, the radii of gyration ($R_G$) and zero-angle intensities $I(0)$ were determined by the PRIMUS program (32) using Guinier's approximation (33) for low $q$ values of all final, subtracted 1D curves: $I(q)=I(0)\exp[-(1/3)q^2R_G^2]$, where $I(q)$ is the scattering intensity at a given scattering vector $q$. $R_G$ and $I(0)$ are extracted by linear fits in a Guinier plot ($\ln[I(q)]$ vs $q^2$), for data ranges with $R_G \cdot q < 1.3$. All parameters extracted from the Guinier approximation are shown in Table S11.

Nominal solvent electron densities ($\rho_{sol}$) for the concentration series were calculated based on the weighed amounts of GdHPDO3A ($C_{17}H_{29}GdN_4O_7$; 558.68 Da; 279 e⁻) and sucrose ($C_{12}H_{22}O_{11}$; 342.30 Da; 182 e⁻) and Milli-Q water ($H_2O$; 18 Da; 10 e⁻) added to prepare the stock solution. The respective volumes were determined by pipetting. Calculated electron densities of samples are shown in Tables S12 to S15. The theoretical protein electron densities ($\rho_p$) were calculated from their respective amino acid sequences (Figure S5), using a range of solvent-excluded volumes $V_i$ of each amino acid $i$ from literature (34): $\rho_p = \sum_i N_i n_i / \sum_i N_i V_i$.

$N_i$ being the number of copies of amino acid $i$ in the sequence, and $n_i$ being the number of electrons per copy of amino acid $i$.

The intensity $I(0)$ of a particle (i.e. protein) with electron density $\rho_p$ is proportional to the product of its concentration $C$ [in mg/mL], and the square of its contrast with respect to the solvent, $\Delta\rho=\rho_p-\rho_{sol}$ (35). Assuming a linear increase of the solvent electron density as a function of contrast agent concentration, the contrast match point (CMP) of a protein can be determined by using a plot of $\sqrt{I(0)/C}$ vs contrast agent concentration (36). The intersection of a linear (or a second-order, i.e. quadratic) fit with the abscissa was used to determine the CMP ($I(0)=0$) for each protein (Figures 1D to 4D).

To check the structural state of each protein, the program CRY SOL (37) was employed to fit the experimental 1D scattering profiles with the theoretical scattering curve from the known protein structures. Both Tp-GH5-Ig (PDB 6TN6) and CaMalDH (PDB 4CL3) structures were thus scored against their respective experimental SAXS data in the absence of contrast agents (0 mM), and at a high concentration of sucrose and GdHPDO3A.

In addition to a comparison with atomic models, *ab initio* shape modeling, using the program DAMMIF from the ATSAS suite (38), was carried out for each reference data (0 mM) and a high concentration of sucrose and GdHPDO3A for both Tp-GH5-Ig and CaMalDH. The underlying pair distance distribution functions $p(r)$, generated from the SAXS data with the program GNOM (39), were also compared within each contrast series.
Results

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Tp-GH5-Ig at 10 mg/mL in GdHPDO3A. A) SAXS curves and Guinier fits (inset). B) CRY SOL fit of crystallographic structure (PDB 6TN6) with SAXS curve at 0 and 723 mM. C) Tp-GH5-Ig R<sub>G</sub> as a function of GdHPDO3A concentration. D) Tp-GH5-Ig I(0) analysis as a function of GdHPDO3A concentration. Determination of the experimental CMP *via* a quadratic fit. Range of theoretical CMPs from amino acid volumes (Table S12).</figcaption></figure>
<figure><img src="image_3.png" /></figure>

A)

Tp-GH5-Ig - Sucrose SAXS curves

<figure><img src="image_4.png" /></figure>

B)

Tp-GH5-Ig - R<sub>G</sub> Analysis

<figure><img src="image_5.png" /></figure>

C)

Tp-GH5-Ig - I(0) Analysis

<figure><img src="image_6.png" /></figure>

D)

**Figure 2.** Tp-GH5-Ig at 6 mg/mL in sucrose. A) SAXS curves and Guinier fits (inset). B) CRY SOL fit of crystallographic structure (PDB 6TN6) with SAXS curve at 0 and 1500 mM sucrose. C) Tp-GH5-Ig R<sub>G</sub> as a function of sucrose concentration. D) Tp-GH5-Ig I(0) analysis as a function of sucrose concentration. Determination of the experimental CMP *via* a linear fit. Range of theoretical CMPs from amino acid volumes (Table S13).
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 3.** CaMalDH at 7.5 mg/mL in GdHPDO3A. A) SAXS curves and Guinier fits (inset). B) CRY SOL fit of crystallographic structure (PDB 4CL3) with SAXS curve at 0 and 495 mM. C) CaMalDH R<sub>G</sub> as a function of GdHPDO3A concentration. D) CaMalDH I(0) analysis as a function of GdHPDO3A concentration. Determination of the experimental CMP *via* a linear fit. Range of theoretical CMPs from amino acid volumes (Table S14).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>CRYSOL model fit of experimental SAXS data for
CaMalDH in the presence of 0 mM and 1000 mM Sucrose</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>CaMalDH - R<sub>G</sub> Analysis</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>CaMalDH - I(0) Analysis</figcaption></figure>

<figure><img src="image_14.png" /></figure>

**Figure 4.** CaMalDH at 4.6 mg/mL in sucrose. A) SAXS curves and Guinier fits (inset). B) CRYSOL fit of crystallographic structure (PDB 4CL3) with SAXS curve at 0 and 1000 mM sucrose. C) CaMalDH R<sub>G</sub> as a function of sucrose concentration. D) CaMalDH I(0) analysis as a function of sucrose concentration. Determination of the experimental CMP *via* a linear fit. Range of theoretical CMPs from amino acid volumes (Table S15).
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 5.** Residual activity measurements of enzymes. A) DNS activity of Tp-GH5-Ig in the presence of GdHPDO3A and Sucrose. B) CaMalDH activity in the presence of GdHPDO3A and sucrose.</figcaption></figure>
## Tp-GH5-Ig and CaMalDH preserve their structural integrity in the presence of sucrose and GdHPDO3A

The similarity and reproducibility of the 1D SAXS curves, as well as the relatively stable values of the radii of gyration ($R_G$) of Tp-GH5-Ig and CaMalDH, indicate that both proteins preserve their conformational and oligomeric states in the presence of sucrose and GdHPDO3A, up to the highest measured concentrations (Figures 1 to 4, panels A and C). The solution structural states of both Tp-GH5-Ig (monomer) and CaMalDH (tetramer) can be very adequately described by their deposited atomic models (PDB IDs 6TN6 and 4CL3, respectively), both the absence of contrast agents, as well as at high contrast agent concentrations (see CRY SOL fits, Figures 1 to 4, panels B). Furthermore, the relative stability of the p(r) functions within each contrast series, as well as the similar *ab intio* shapes, indicate that no major conformational changes, detectable at low resolution, occur as a function of contrast agent concentration for either of the proteins (Fig. S6).

The $R_G$ of Tp-GH5-Ig in GdHPDO3A (Fig. 1C), and CaMalDH in sucrose (Fig. 4C), remain constant, within random variations, as a function of contrast agent concentration. A single outlier is observed at the highest concentration in both cases, probably due to noisy data and parasitic scattering at the lowest q-values, which required also a modification of the respective Guinier fit ranges. In the case of Tp-GH5-Ig in sucrose (Fig. 2C), and of CaMalDH in GdHPDO3A (Fig. 3C), there is a slight tendency of the $R_{GS}$ to increase and decrease, respectively, as a function of contrast agent concentration. These tendencies are moderate (<3% and <6%, respectively, Table S11), apart from individual outliers at the highest concentrations. This minor, systematic, evolution of the experimental $R_{GS}$ could be due to solvent effects (20, 23), or to a modification of the inter-particle repulsion, which is present for both proteins at the concentrations used in the present study (Figures S7 and S8).

The square root of the forward scattered intensities, $\sqrt{I}(0)$, varied in two cases strictly linearly with the contrast agent concentration (Figures 2 to 4, panels D), as expected for a monodisperse solution of particles with identical, constant, chemical composition and stable oligomeric state (36). In the case of sucrose, there are excellent matches between the experimentally determined contrast match points (CMPs, i.e. $I(0)=0$) of Tp-GH5-Ig and CaMalDH (Figures 2, 4 panels D, and Tables S13 and S15, respectively), and the ones calculated from their amino acid residue sequences (Figure S5). The experimental CMPs of both proteins in GdHPDO3A are inferior to the theoretical ranges, predicted from their sequences and amino acid volumes reported in literature (Figures 1 and 3, panels D, and Tables S12 and S14). Moreover, the $\sqrt{I}(0)$ of Tp-GH5-Ig do not display a linear behavior as a function of GdHPDO3A concentration. Both phenomena have been interpreted in terms of a combination of specific interactions of the contrast agents with proteins, and unspecific solvent effects (23), and are not necessarily an indication of structural alterations or changes of the oligomeric state of proteins.

Taken together, the ensemble of the SAXS data in the present study suggest that both Tp-GH5-Ig and CaMalDH conserve both their global structural conformations and oligomeric states, up to the highest concentrations of sucrose and GdHPDO3A probed in our study (Fig. 1-4, panels B).

## Long incubation times at high concentrations of sucrose and GdHPDO3A do not affect the enzymatic activities of Tp-GH5-Ig and CaMalDH

Contrast agents such as sucrose and lanthanide complexes typically occupy volume fractions of 20-40% in solution at molar concentrations (24), and thus represent physico-chemical
environments which can be very distinct from dilute, aqueous buffer conditions which are usually employed in BioSAXS experiments. It is therefore important to assess if the function of biomacromolecules are impacted as a consequence of prolonged (i.e. minutes to hours, as required for SAXS experiments) incubation times to high concentrations of such contrast agents.

In addition to the structural analyses of the protein structures, we therefore monitored the residual enzymatic activity of the endo-β-1,4-mannanase and malate dehydrogenase after several hours of incubation to various concentrations of sucrose and GdHPDO3A (see Material and Methods). This method is routinely applied to compare the effects of a chemical-physical treatment on the stability and functional integrity of a given enzyme, in particular if the enzymatic test is easy to record and allows to monitor a large spectrum of conditions, as exemplified by studies on MalDHs and other enzymes (40–46). The results show that for both enzymes studied in this work, no significant variation of the residual enzymatic activity is observed in the range from 0 to 1M sucrose or GdHPDO3A, and exposure times up to 24 hours (Figure 5).

# Discussion

## Sucrose and GdHPDO3A do not impact the structural integrity of Tp-GH5-Ig and CaMalDH

The structural integrity of both proteins in the presence of contrast agents was evaluated by the following parameters: radii of gyration ($R_G$), forward scattered intensities ($I(0)$), and contrast match-points (CMPs). The fits of atomic models in aqueous buffers (i.e. in the absence of contrast agents) were used as references.

CRYSOL fits of the PDB models 6TN6 and 4CL3 with the respective SAXS data in aqueous buffers were very good, thus validating the atomic models and the oligomeric states of both proteins: monomeric for Tp-GH5-Ig, and tetrameric for CaMalDH (Figures 1 to 4, panels B). The relatively high values of the $\chi^2$ fits in the absence of contrast agents (0 mM) can be explained by inter-particle effects present at the elevated concentrations employed here (Tp-GH5-Ig: 6 mg/mL, CaMalDH: 10 mg/mL), in order to improve signal/noise in the contrast series. These concentrations do not correspond to dilute solution conditions and discrepancies between the theoretical curves and the experimental data are mainly present at the lowest angles, thus increasing the $\chi^2$ fit values artificially. Indeed, repulsive inter-particle effects were observed for both proteins, as a function of their concentrations, in the absence of contrast agents (Fig. S7 and S8). However, significant changes of the protein oligomeric states, as well as the overall conformations can be ruled out in our case: the atomic models of both proteins, which fit the SAXS data in the absence of contrast agents (0 mM), fit also SAXS data at higher contrast agent concentrations excellently, with $\chi^2$ values close to 1 (Figures 1 to 4, panel B).

The $R_G$ of both proteins evolved only moderately by 1-2% in most cases with the contrast agent concentration, when excluding the highest concentration data point in each series (Figures 1, 2 and 4, panels C; Table S11). A notable exception was the GdHPDO3A series of CaMalDH where the $R_G$ increased by 6%, excluding the highest concentration (Fig. 3B; Table S11). The systematic outliers observed at the highest contrast agent concentrations in all cases (Fig. 1 to 4, panels C) are probably due to subtraction issues at low angles, related to parasitic scattering, capillary fouling, and a weak signal/noise (see Fig. S1 to S4). While significant, the relative changes of the $R_G$ values observed here are comparable, or inferior, to those due
to repulsive inter-particle effects, observed as a function of protein concentrations in the absence of contrast agents (Fig. S7 and S8). (Tp-GH5-Ig and CaMalDH have pI values of 4.93 and 5.16, respectively). The continuous increase of the protein RGS in the GdHPDO3A series (Fig. 1C and 3C) could therefore be due to the free GdHPDO3A in solution screening, at least partially, the repulsive protein-protein interactions.

An alternative explanation of a continuous evolution of the protein RGS could be preferential interactions of the contrast agents with the proteins. SAXS and SANS data on solubilized proteins have indeed shown that the solvent properties in the vicinity of their surfaces can be altered with respect to the bulk solvent (47, 48), and that the modifications depend on the physico-chemical properties of proteins, in particular the surface charge distribution of amino acids (49, 50). Surface charges can result in the presence of ion clouds for nucleic acids, e.g. in protein-DNA complexes (17–19), but also for proteins (51). In CV-SAXS experiments on lysozyme and glucose isomerase in NaCl and KCl, protein-specific increases of the hydration shell density were observed (52). Finally, sucrose and sucralose have been shown to display preferential interaction with the protein myoglobin (53), resulting in variations of the RG of 5-10% at molar concentrations, which is in agreement with the relative changes of RGS found in our study.

The square root of the forward scattered intensity of a particle in solution, $\sqrt{I}(0)$, is proportional to the electron density difference of the particle with respect to the bulk solvent ($\Delta\rho$), integrated over the solvent-excluded volume of the particle (35). This volume can include solvent in the vicinity of the particle if it has a different scattering length density than the bulk solvent. A linear behavior of $\sqrt{I}(0)$ as a function of contrast agent concentration is observed in most cases in our study, up to the highest concentrations measured (Figures 2 to 4, panels D). Assuming that the bulk solvent electron density increases linearly with the contrast agent concentration, the linear behavior of $\sqrt{I}(0)$ implies that solvent-excluded volumes of the solubilized proteins are constant. In other terms, there is no major evolution of their oligomeric states, i.e. a significant population of aggregated or dissociated species.

A slight deviation from a linear behavior of $\sqrt{I}(0)$ is observed for one of the conditions studied here, i.e. Tp-GH5-Ig in GdHPDO3A (Figure 1D). Assuming again that the bulk solvent electron density increases linearly with the contrast agent concentration, such a behavior can indicate a variable solvent-excluded volume of the protein, or a change of its electron density (or a combination of both). In both cases, “volume of the protein” includes its hydration shell. Deviations from non-linear behavior of $\sqrt{I}(0)$ have been observed for sucrose/sucralose (53) and for iohexol (23) as co-solutes in CV-SAXS experiments on proteins.

In the latter case, the effect has been interpreted in terms of preferential interactions of a few iohexol molecules with the protein (P1, 112 kDa) surface, and a decrease of the global density of a 3 Å hydration shell by 20% with respect to the bulk (validated by CRY SOL). This decrease of the hydration shell density was compatible with a 5% decrease of the experimentally measured CMP, with respect to the one expected from the protein's amino acid sequence.

In the present work, a comparable decrease of the experimentally measured CMPs with respect to the theoretically predicted ones (by about 7-8%) for Tp-GH5-Ig (53 kDa) and CaMalDH (144 kDa) was observed for GdHPDO3A. It could thus quantitatively be explained by a similar decrease of the global hydration shell density. Differences in the numerical values of the mismatches of experimental and theoretical CMPs for iohexol and GdHPDO3A might be due to the nature of the protein sizes and surfaces, as well as the different contrast agent concentrations: 600 mM iohexol in the previous study, and > 1000 mM GdHPDO3A in the present study.

For $\sqrt{I}(0)=0$, i.e. the contrast-match point (CMP), the integral of $\Delta\rho$ over the solvent-excluded volume is zero. Knowing the chemical composition of a given protein, as well as its solvent-
excluded volume from its amino acid composition (34, 36), the theoretically predicted CMP can be compared with the experimental one. In our study the results of these comparisons are shown in Figures 1-4, panels D, and Tables S12-S15. In the sucrose series, we obtain a good to excellent agreement between the range of theoretically predicted CMPs and the experimental ones for both proteins (Figures 2 and 4, panels D; Tables S13 and S15). In both GdHPDO3A series, the experimental match-points are significantly below the range of theoretically predicted ones (Figures 1 and 3, panels D; Tables S12 and S14). A lower CMP in CV-SAXS experiments can occur if the contrast molecules are preferentially excluded from the hydration shell as has been observed for iohexol (24).

From the ensemble of our SAXS data, we conclude that the minor evolutions of the R<sub>G</sub>, deviations from non-linear behavior of √I(0), and mis-matches between experimental and theoretical CMPs in some of the cases can be explained by the following effects: preferential solvent interaction, screening of repulsive inter-particle interactions, and systematic errors of buffer subtractions at lowest angles, in particular at high contrast agent concentrations.

**Prolonged exposure to high concentrations of sucrose and GdHPDO3A do not diminish the enzymatic activity of Tp-GH5-Ig and CaMalDH**

The absence of a loss of activity at high concentrations shows that both compounds may be considered as neutral, i.e. they do not alter the enzyme functionality when tested in the standard assay. Here, neutrality refers to the concept based on salt effects: indeed, after incubation in the presence of growing concentration of a salt, when residual activity measurement of a given enzyme shows a loss of activity it may be due to effects on conformational stability and association-dissociation equilibria that allowed a classification (54, 55). Those that dissociate and unfold proteins are considered as salting-in salt, in contrast, salting-out salt favors loss of solubility and precipitation. In this framework, a solute is considered neutral if it does not induce detectable structural or functional changes in the protein, even at high concentrations.

Endo-β-1,4-mannanase is a glycoside hydrolase that cleave the β-1,4-glycosidic bonds in mannan and related polysaccharides like galactomannan. Its mechanism involves active site residues that promote the binding and hydrolysis of these specific carbohydrate structures. Indeed, the active-site cavity of Tp-GH5-Ig contains the catalytic acid/base residue E198 and the nucleophile E317, along with several aromatic residues (25).

Malate dehydrogenase is a well-characterized enzyme present in all organisms (56, 57). It operates in the tricarboxylic acid cycle, in which it catalyzes the interconversion of oxaloacetate and malate linked to the oxidation/reduction of dinucleotide coenzymes. As is the case with all MalDHs, three conserved arginine residues (R82, R88 and R151) anchor the substrate within the catalytic vacuole in CaMalDH during the catalytic cycle. Then, H175 and D148, which form a proton relay system, promote the transfer of a hydride ion from the pro-R face of NADH to oxaloacetate to produce malate.

These enzymatic mechanisms are often finely tuned and sensitive to alterations in the immediate active site environment. Even subtle interactions or changes in solvent properties, caused by the addition of a solute, could potentially influence the precise positioning and protonation states of the catalytic residues, or affect substrate binding and transition state stabilization. Therefore, a neutral solute would suggest that it either does not access the enzyme's microenvironment to any significant extent or that its presence does not perturb the delicate balance of forces and interactions crucial for the catalytic cycle of each enzyme under the experimental conditions tested here. This lack of observable effect on proteins structure and function is particularly noteworthy given that high solute concentrations can often lead to non-specific effects on enzyme activity.
Due to several constraints imposed by the enzymatic protocols (e.g. mixture of reaction agents and buffers, necessity to dilute the protein concentrations to an optimal range), it was not possible to measure the residual activities in the exact same conditions as those used in the SAXS experiments. It was in particular necessary to dilute the protein concentrations by about a factor of 7-15 or 60-100 for the residual activity measurements, with respect to the SAXS conditions, for endo-β-1,4-mannanase and malate dehydrogenase, respectively. However, these conditions resulted in an actual increase of the stoichiometric ratio of contrast agents per protein solubilized by the same factor in each case (Tables S16 and S17). In this sense, the incubation conditions for the enzymatic assays can be considered as even more “extreme” than the SAXS conditions, and thus validate the neutrality of both contrast agents on the functional properties of both proteins after long exposure times to them.

## Limitations, choice of contrast agents, and perspectives of CV-SAXS

In the present study and previous work (23, 24) we showed that new classes of contrast molecules, i.e. heavy-atom based complexes, can readily achieve solvent electron densities superior to sucrose. Importantly, in the present study we show that both for a “classical” contrast agent such as sucrose, as well as for the lanthanide-based GdHPDO3A complex, the structural integrity and enzymatic activity is preserved for two specific proteins, Tp-GH5-Ig and CaMalDH. To the best of our knowledge, this is the first time that such a study has been undertaken in a CV-SAXS experiment.

While these results are encouraging for a broader use of CV-SAXS for structural biology of biomacromolecular complexes in solution, there are also limitations. Caution is therefore required before it can be generalized and applied to diverse biological systems (e.g. protein-RNA/DNA and membrane protein complexes).

SAXS contrast variation has currently several general drawbacks with respect to its sister technique SANS: 1) a limited range of solvent electron densities, which does not cover all biomolecules (in particular RNA/DNA), 2) potential interference of contrast agents with the structural integrity of biomacromolecules and assemblies, 3) potential inter-particle effects and preferential interaction, and 4) limited possibilities to change the scattering properties of the biomacromolecules (which can be achieved by deuteration in SANS).

Concrete examples of these limitations in the present study include the inter-particle effects and preferential interactions of the contrast agents with the proteins, which result in shifts of the contrast-match points (CMP) with respect to theoretically expected values, as well as a non-linear behavior of $\sqrt{I(0)}$ in one case.

Such effects, potentially related to modifications of the buffer properties (influencing inter-particle interactions) and/or specific interactions with protein surfaces and hydration shells, are difficult to predict from first principles. Even though co-crystallization and NMR paramagnetic relaxation (58) can indicate such interactions in some cases, more experimental CV-SAXS data on a broad variety of biological systems and buffer systems is required to better understand these phenomena.

As in SANS (where macromolecular deuteration and/or H₂O/D₂O exchange can lead to oligomerization/aggregation (59)), the structural integrity of a given biomacromolecule (or an assembly) has thus to be verified case-by-case in CV-SAXS, as a function of the contrast agent and its concentration. A detailed I(0) and CMP analysis, as presented here, should therefore be routinely carried out. Moreover, if possible, the biomacromolecule should be measured at variable concentrations at each contrast condition, in order to evaluate potential inter-particle effects.

Table 1 summarizes the pros and cons of both contrast molecules used in the present study, as well as reports from literature which have used either of them.
<table><caption>**Table 1. Pros and Cons of the two contrast agents used in the present study.** This table summarizes the pros and cons of sucrose and GdHPDO3A used in the present study, as well as observations reported in the literature using either of the molecules.</caption><thead><tr><th></th><th>Strengths (+)</th><th>Weaknesses (-)</th></tr></thead><tbody><tr><td>Sucrose</td><td><ul><li>cheap ($)</li><li>weak absorption of X-rays</li><li>preserves structural integrity</li></ul></td><td><ul><li>limited solvent electron density</li><li>possible preferential interaction</li><li>contrast properties cannot be improved</li><li>difficult to lyophilize</li><li>lower protein concentrations in final mixture</li></ul></td></tr><tr><td>GdHPDO3A</td><td><ul><li>high solvent electron density</li><li>preserves structural integrity</li><li>contrast properties can be improved</li><li>easy to lyophilize</li><li>higher protein concentrations in final mixture</li></ul></td><td><ul><li>expensive ($$$)</li><li>high absorption of X-rays</li><li>possible preferential interaction</li><li>possible inter-particle effects</li></ul></td></tr></tbody></table>

While CV-SAXS has some limitations, only a few contrast molecules have been explored to date. Further promising developments can thus be expected in the future. In the case of Lanthanide-based compounds, variations could include changing the cages of the complexes, the central heavy atom and other properties. Such modifications could potentially result in even higher solvent electron densities than those reported in the current study, and different interaction with biomolecules. Together with the availability of a dense network of SAXS instruments (synchrotron beamlines or home sources) and their easy use, CV-SAXS should become more broadly available to a larger user community.

The equivalent of perdeuteration in SANS, i.e. a homogeneous and complete modification of the electron densities of biological molecules seems impossible to realize in SAXS, at least without a complete alteration of the biological function. However, several ingenious strategies have been developed to alter contrast of specific parts of biological and soft matter systems for X-rays:

In some soft matter systems, the scattering properties and contrast have been changed by the replacement of specific ions. A prominent example is the use of cesium ions, instead of the usual sodium ions, in sodium dodecyl sulfate (SDS) micelles, which allows a strong modulation of contrast (60–62). A replacement of sodium ion with a cesium ion was also used to probe the assembly two specific surfactants, one zwitterionic (oleyl amidopropyl betaine) and the other anionic (Aerosol-OT) by CV-SAXS using several salt solutions as contrast agents (63).

In the case of a calmodulin-peptide complex, the two Ca²⁺ ion of the protein were replaced by Pd²⁺ ion in order to increase contrast and obtain a pair-distance distribution between the heavy atoms by CV-SAXS in 65% sucrose, where the protein and peptide signals were matched on average (64). Distance restraints in DNA strands were equally obtained by SAXS using gold nanoparticles as molecular rulers, based on the increased local electron density (65), or on the anomalous scattering (ASAXS) properties of gold (66). Anomalous scattering of non-
canonical amino acids containing bromine was also used as a molecular ruler to determine
distance restraints by ASAXS from intrinsically disordered proteins (IDPs) (67).

Conclusions

In the present work, we have probed the structural integrity, as well as the residual enzymatic activity, of two proteins, Tp-GH5-Ig and CaMalDH, in the presence of a broad concentration range of sucrose and GdHPDO3A. The ensemble of our data suggest that both proteins preserve their structural integrity, conformation and oligomeric state in the presence of both contrast agents, and up to the highest measured concentrations. Moreover, our data show that their function is not significantly perturbed after long time exposure with the contrast agent. These results are encouraging to further pursue methodological developments in contrast variation SAXS, in particular testing and screening a broad variety of new contrast molecules.

Author contributions

VMS performed research, conducted experiments, analyzed data and wrote the manuscript. AT conducted experiments and analyzed data. DM performed research and contributed the malate dehydrogenase system. BF performed research. WG performed research and contributed the endo-β-1,4-mannanase system. JP performed research. EG designed and performed research. FG designed and performed research, conducted experiments, analyzed data, and wrote the manuscript.

Declaration of interest

The authors declare no competing interests.

Acknowledgements

This work was supported by the French ANR "DeepSAXS" grant (ANR-21-CE11-0025). We would like to express our gratitude to SOLEIL synchrotron for the opportunity to use SWING beamline under BAG proposal numbers 20210859 and 20231127, and in-house proposal number 99210123.

References

1. 2022. Small angle scattering: Part A: Methods for structural investigation. First edition. Cambridge, MA San Diego, CA Oxford London: Academic Press.

2. 2023. Small angle scattering. part B: Methods for structural interpretation. First edition. Cambridge, MA San Diego, CA Oxford London: Academic Press, an Imprint of Elsevier.
3. Chari, A., and H. Stark. 2023. Prospects and Limitations of High-Resolution Single-Particle Cryo-Electron Microscopy. *Annu. Rev. Biophys.* 52:391–411.

4. Bertoline, L.M.F., A.N. Lima, J.E. Krieger, and S.K. Teixeira. 2023. Before and after AlphaFold2: An overview of protein structure prediction. *Front. Bioinform.* 3:1120370.

5. Agarwal, V., and A.C. McShan. 2024. The power and pitfalls of AlphaFold2 for structure prediction beyond rigid globular proteins. *Nat Chem Biol.* 20:950–959.

6. Jeffries, C.M., J. Ilavsky, A. Martel, S. Hinrichs, A. Meyer, J.S. Pedersen, A.V. Sokolova, and D.I. Svergun. 2021. Small-angle X-ray and neutron scattering. *Nature Reviews Methods Primers.* 1:70.

7. Trewhella, J. 2022. Recent advances in small-angle scattering and its expanding impact in structural biology. *Structure.* 30:15–23.

8. Brosey, C.A., and J.A. Tainer. 2019. Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology. *Current Opinion in Structural Biology.* 58:197–213.

9. Gabel, F. 2015. Small-Angle Neutron Scattering for Structural Biology of Protein-RNA Complexes. *Methods Enzymol.* 558:391–415.

10. Mahieu, E., and F. Gabel. 2018. Biological small-angle neutron scattering: recent results and development. *Acta Crystallographica Section D.* 74:715–726.

11. Haertlein, M., M. Moulin, J.M. Devos, V. Laux, O. Dunne, and V. Trevor Forsyth. 2016. Chapter Five - Biomolecular Deuteration for Neutron Structural Biology and Dynamics. In: Kelman Z, editor. Methods in Enzymology. Academic Press. pp. 113–157.

12. Bayrak, M., Q. Han, T.L. Greaves, S. Seibt, H. Yu, J. Floury, J. Mata, A. Logan, and C.E. Conn. 2024. Correlating structure and activity of pepsin enzyme in H2O and D2O for the study of gastric digestion. *Food Chemistry Advances.* 4:100638.

13. Bolze, J., K.D. Hoerner, and M. Ballauff. 1996. Adsorption of the Nonionic Surfactant Triton X-405 on Polysterene Latex Particles as Monitored by Small-Angle X-ray Scattering. *Langmuir.* 12:2906–2912.

14. Kiselev, A.M., P. Lesieur, A.M. Kisselev, D. Lombardo, M. Killany, and S. Lesieur. 2001. Sucrose solutions as prospective medium to study the vesicle structure: SAXS and SANS study. *Journal of Alloys and Compounds.* 328:71–76.

15. Ballauff, M. 2011. Analysis of Polymer Colloids by Small-Angle X-Ray and Neutron Scattering: Contrast Variation. *Advanced Engineering Materials.* 13:793–802.

16. Luzzati, V., A. Tardieu, and L.P. Aggerbeck. 1979. Structure of serum low-density lipoprotein: I. A solution X-ray scattering study of a hyperlipidemic monkey low-density lipoprotein. *Journal of Molecular Biology.* 131:435–473.

17. Pollack, L. 2011. SAXS Studies of Ion–Nucleic Acid Interactions. *Annual Review of Biophysics.* 40:225–242.

18. San Emeterio, J., S.A. Pabit, and L. Pollack. 2022. Chapter Two - Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis. In: Tainer JA, editor. Methods in Enzymology. Academic Press. pp. 41–83.

19. Tokuda, J.M., S.A. Pabit, and L. Pollack. 2016. Protein-DNA and ion-DNA interactions revealed through contrast variation SAXS. *Biophys Rev.* 8:139–149.

20. Schneidman-Duhovny, D., M. Hammel, J.A. Tainer, and A. Sali. 2013. Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments. *Biophysical Journal.* 105:962–974.
21. Jeffries, C.M., M.A. Graewert, C.E. Blanchet, D.B. Langley, A.E. Whitten, and D.I. Svergun. 2016. Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments. *Nat Protoc.* 11:2122–2153.

22. Hutin, S., A. Guillotin, C. Zubieta, and M.D. Tully. 2023. Chapter Six - Structural characterization of protein–DNA complexes using small angle X-ray scattering (SAXS) with contrast variation. In: Jez J, editor. *Methods in Enzymology*. Academic Press. pp. 163–194.

23. Gabel, F., S. Engilberge, E. Schmitt, A. Thureau, Y. Mechulam, J. Pérez, and E. Girard. 2022. Medical contrast agents as promising tools for biomacromolecular SAXS experiments. *Acta Crystallogr D Struct Biol.* 78:1120–1130.

24. Gabel, F., S. Engilberge, J. Perez, and E. Girard. 2019. Medical contrast media as possible tools for SAXS contrast variation. *IUCrJ.* 6:521–525.

25. Da Silva, V.M., A.D. Cabral, M.A. Sperança, F.M. Squina, J.R.C. Muniz, L. Martin, Y. Nicolet, and W. Garcia. 2020. High-resolution structure of a modular hyperthermostable endo-β-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain. *Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics.* 1868:140437.

26. Talon, R., N. Coquelle, D. Madern, and E. Girard. 2014. An experimental point of view on hydration/solvation in halophilic proteins. *Front. Microbiol.* 5.

27. Gasteiger, E., C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel, and A. Bairoch. 2005. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. *The Proteomics Protocols Handbook*. Totowa, NJ: Humana Press. pp. 571–607.

28. Miller, G.L. 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. *Anal. Chem.* 31:426–428.

29. Thureau, A., P. Roblin, and J. Perez. 2021. BioSAXS on the SWING beamline at Synchrotron SOLEIL. *Journal of Applied Crystallography.* 54:1698–1710.

30. Munoz, L., Y.-M. Abiven, M.-E. Couprie, M. Valléau, A. Noureddine, J. Pérez, A. Thureau, and F. Saint-Aubin. 2023. Robotic Process Automation: on the Continuity of Applications Development at SOLEIL. p. THMBCMO32.

31. Girardot, R., G. Viguier, J. Pérez, and M. Ounsy. 2015. FOXTROT: A JAVA-based application to reduce and analyse SAXS and WAXS piles of 2D data at synchrotron SOLEIL. pp. 14–16.

32. Manalastas-Cantos, K., P.V. Konarev, N.R. Hajizadeh, A.G. Kikhney, M.V. Petoukhov, D.S. Molodenskiy, A. Panjkovich, H.D.T. Mertens, A. Gruzinov, C. Borges, C.M. Jeffries, D.I. Svergun, and D. Franke. 2021. *ATSAS 3.0*: expanded functionality and new tools for small-angle scattering data analysis. *J Appl Crystallogr.* 54:343–355.

33. Guinier, A., and G. Fournet. 1955. Small Angle Scattering of X-rays. New York: John Wiley & Sons.

34. PERKINS, S.J. 1986. Protein volumes and hydration effects. *European Journal of Biochemistry.* 157:169–180.

35. Svergun, D.I., and M.H.J. Koch. 2002. Small-angle scattering studies of biological macromolecules in solution. *Reports on Progress in Physics.* 66:1735–1782.

36. Bernard Jacrot. 1976. The study of biological structures by neurtron scattering from solution. *Reports on Progress in Physics.* 39:911–953.

37. Dmitri I. Svergun, C. Barberato, and Michel H. J. Koch. 1995. CRYSOL - a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates. *Journal of Applied Crystallography.* 28:768–773.
38. Franke, D., and D.I. Svergun. 2009. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. *J. Appl. Cryst.* 42:342–346.

39. Dmitri I. Svergun. 1992. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. *Journal of Applied Crystallography*. 25:495-503.

40. Madern, D., M. Camacho, A. Rodríguez-Arnedo, M.-J. Bonete, and G. Zaccai. 2004. Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii. *Extremophiles*. 8:377-384.

41. Ohkuri, T., and A. Yamagishi. 2007. The Effects of Mutations at Position 253 on the Thermostability of the Bacillus subtilis 3-Isopropylmalate Dehydrogenase Subunit Interface. *The Journal of Biochemistry*. 141:791-797.

42. Coquelle, N., E. Fioravanti, M. Weik, F. Vellieux, and D. Madern. 2007. Activity, Stability and Structural Studies of Lactate Dehydrogenases Adapted to Extreme Thermal Environments. *Journal of Molecular Biology*. 374:547-562.

43. Rahimzadeh, M., K. Khajeh, M. Mirshahi, M. Khayatian, and R. Schwarzenbacher. 2012. Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase. *International Journal of Biological Macromolecules*. 50:1175-1182.

44. Delgado, Y., M. Morales-Cruz, J. Hernández-Román, Y. Martínez, and K. Griebenow. 2014. Chemical glycosylation of cytochrome c improves physical and chemical protein stability. *BMC Biochemistry*. 15:16.

45. Blanquart, S., M. Groussin, A. Le Roy, G.J. Szöllosi, E. Girard, B. Franzetti, M. Gouy, and D. Madern. 2021. Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties. *Molecular Biology and Evolution*. 38:3754-3774.

46. Madern, D., F. Halgand, C. Houée-Levin, A.-B. Dufour, S. Coquille, S. Ansanay-Alex, S. Sacquin-Mora, and C. Brochier-Armanet. 2024. The Characterization of Ancient Methanococcales Malate Dehydrogenases Reveals That Strong Thermal Stability Prevents Unfolding Under Intense γ-Irradiation. *Molecular Biology and Evolution*. 41:msae231.

47. Svergun, D.I., S. Richard, M.H.J. Koch, Z. Sayers, S. Kuprin, and G. Zaccai. 1998. Protein hydration in solution: experimental observation by x-ray and neutron scattering. *Proceedings of the National Academy of Sciences USA*. 95:2267-2272.

48. Zhang, F., F. Roosen-Runge, M.W.A. Skoda, R.M.J. Jacobs, M. Wolf, P. Callow, H. Frielinghaus, V. Pipich, S. Prevost, and F. Schreiber. 2012. Hydration and interactions in protein solutions containing concentrated electrolytes studied by small-angle scattering. *Physical Chemistry Chemical Physics*. 14:2483-2493.

49. Kim, H.S., A. Martel, E. Girard, M. Moulin, M. Härtlein, D. Madern, M. Blackledge, B. Franzetti, and F. Gabel. 2016. SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell. *Biophysical Journal*. 110:2185-2194.

50. Linse, J.-B., and J.S. Hub. 2023. Scrutinizing the protein hydration shell from molecular dynamics simulations against consensus small-angle scattering data. *Communications Chemistry*. 6:272.

51. Ivanović, M.T., L.K. Bruetzel, R. Shevchuk, J. Lipfert, and J.S. Hub. 2018. Quantifying the influence of the ion cloud on SAXS profiles of charged proteins. *Phys. Chem. Chem. Phys.* 20:26351–26361.

52. Schneidman-Duhovny, D., M. Hammel, J.A. Tainer, and A. Sali. 2013. Accurate SAXS profile computation and its assessment by contrast variation experiments. *Biophys J*. 105:962–74.

53. Kuroiwa, I., Y. Maki, K. Matsuo, and M. Annaka. 2024. Protein Preferential Solvation in (Sucralose + Water) Mixtures. *J. Phys. Chem. B*. 128:676–683.
54. Von Hippel, P.H., and T. Schleich. 1969. Ion effects on the solution structure of biological macromolecules. *Accounts of Chemical Research*. 2:257-265.

55. Arakawa, T., R. Bhat, and S.N. Timasheff. 1990. Preferential interactions determine protein solubility in three-component solutions: the magnesium chloride system. *Biochemistry*. 29:1914-1923.

56. Brochier-Armanet, C., and D. Madern. 2021. Phylogenetics and biochemistry elucidate the evolutionary link between l-malate and l-lactate dehydrogenases and disclose an intermediate group of sequences with mix functional properties. *Biochimie*. 191:140-153.

57. Coquille, S., C.S. Pereira, J. Roche, G. Santoni, S. Engilberge, C. Brochier-Armanet, E. Girard, F. Sterpone, and D. Madern. 2025. Allotery and Evolution: A Molecular Journey Through the Structural and Dynamical Landscape of an Enzyme Super Family. *Molecular Biology and Evolution*. 42:msae265.

58. Madl, T., T. Güttler, D. Görlich, and M. Sattler. 2011. Structural Analysis of Large Protein Complexes Using Solvent Paramagnetic Relaxation Enhancements. *Angewandte Chemie International Edition*. 50:3993-3997.

59. Giubertoni, G., M. Bonn, and S. Woutersen. 2023. D2O as an Imperfect Replacement for H2O: Problem or Opportunity for Protein Research? *J. Phys. Chem. B*. 127:8086-8094.

60. Vass, Sz., J. Pleštil, P. Laggner, T. Gilányi, S. Borbély, M. Kriechbaum, Gy. Jákli, Z. Décsy, and P.M. Abuja. 2003. Models of Micellar Structure Tested by SANS and SAXS (from a Kratky Camera) in Cesium Dodecyl Sulfate Solution. *J. Phys. Chem. B*. 107:12752-12761.

61. Kanungo, S., J. Narayanan, V.K. Aswal, J.R. Bellare, and P.S. Goyal. 2012. Study of core-shell structure of cesium dodecylsulfate micelles using small-angle X-ray and neutron scattering. *Journal of Molecular Liquids*. 175:38-43.

62. Lee, H.S., M. Adhimoolam Arunagirinathan, A. Vagias, S. Lee, J.R. Bellare, H.T. Davis, E.W. Kaler, A.V. McCormick, and F.S. Bates. 2014. Almost Fooled Again: New Insights into Cesium Dodecyl Sulfate Micelle Structures. *Langmuir*. 30:12743-12747.

63. McCoy, T.M., J.B. Marlow, A.J. Armstrong, A.J. Clulow, C.J. Garvey, M. Manohar, T.A. Darwish, B.J. Boyd, A.F. Routh, and R.F. Tabor. 2020. Spontaneous Self-Assembly of Thermoresponsive Vesicles Using a Zwitterionic and an Anionic Surfactant. *Biomacromolecules*. 21:4569-4576.

64. Grishaev, A., N.J. Anthis, and G.M. Clore. 2012. Contrast-Matched Small-Angle X-ray Scattering from a Heavy-Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin-Peptide Complex. *J. Am. Chem. Soc*. 134:14686-14689.

65. Zettl, T., R.S. Mathew, X. Shi, S. Doniach, D. Herschlag, P.A.B. Harbury, and J. Lipfert. 2018. Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions. *Science Advances*. 4:eaar4418.

66. Zettl, T., R.S. Mathew, S. Seifert, S. Doniach, P.A.B. Harbury, and J. Lipfert. 2016. Absolute Intramolecular Distance Measurements with Angstrom-Resolution Using Anomalous Small-Angle X-ray Scattering. *Nano Letters*. 16:5353-5357.

67. Yu, M., A.Yu. Gruzinov, H. Ruan, T. Scheidt, A. Chowdhury, S. Giofrè, A.S.A. Mohammed, J. Caria, P.F. Sauter, D.I. Svergun, and E.A. Lemke. 2024. A genetically encoded anomalous SAXS ruler to probe the dimensions of intrinsically disordered proteins. *Proceedings of the National Academy of Sciences*. 121:e2415220121.