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. Author manuscript; available in PMC: 2013 Jul 17.
Published in final edited form as: Methods Mol Biol. 2012;922:101–122. doi: 10.1007/978-1-62703-032-8_6

Sample Preparation Methods to Analyze DNA-Induced Structural Changes in Replication Protein A

Chris A Brosey, Susan E Tsutakawa, Walter J Chazin
PMCID: PMC3713622  NIHMSID: NIHMS485626  PMID: 22976179

Abstract

Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding protein (SSB) in eukaryotes, serves to protect ssDNA generated during these events and to recruit and organize other DNA-processing factors requiring access to ssDNA substrates. RPA engages ssDNA in distinct, progressive binding modes, which are thought to correspond to different functional states of the protein during the course of DNA processing. Structural characterization of these unique complexes has remained challenging, however, as RPA is a multi-domain protein characterized by a flexible, modular organization. Biophysical approaches that are well suited to probing time-varying architectures, such as NMR and small-angle X-ray and neutron scattering (SAXS/SANS), when integrated with computational methods, can provide critical insights into the architectural changes associated with RPA’s different DNA-binding modes. The success of these methods, however, is highly contingent upon the purity, homogeneity, and stability of the sample under study. Here we describe a basic protocol for characterizing and optimizing sample conditions for RPA/ssDNA complexes prior to study by SAXS and/or SANS.

Keywords: Replication protein A, Single-stranded DNA-binding protein, Oligonucleotide-/oligosaccharide-binding fold, DNA processing, Protein modularity, Solubility screening, Small-angle X-ray scattering, Small-angle neutron scattering, Size-exclusion chromatography, Multi-angle light scattering

1. Introduction

Every organism requires a ssDNA-binding activity to protect ssDNA from chemical and enzymatic assault, while resolving nucleic acid secondary structure that can derail DNA-processing machinery (1–3). In addition to this critical role, the primary eukaryotic single-stranded DNA-binding protein (SSB), Replication Protein A (RPA), also serves as a central scaffold for the regulated assembly, exchange and disassembly of multi-protein DNA-processing complexes at ssDNA substrates. A heterotrimer, RPA consists of three polypeptide subunits (RPA70, RPA32, RPA14), which contain a total of seven globular domains connected by flexible linkers and a single disordered domain. Three of these domains (70C, 32D, 14) interface through a hydrophobic three-helix bundle to form the trimeric core of RPA from which emanate the N-terminal domains of RPA70 (70B, 70A, 70N), the C-terminal domain of RPA32, and the disordered N-terminus of RPA32 (32N) (4). As with the majority of SSBs, all domains are oligonucleotide-/oligosaccharide-binding (OB) folds with the exception of RPA32C (a winged helix domain). All RPA domains have been characterized individually or in tandem at high structural resolution (5–11). The high-affinity ssDNA binding (Kd 10 −9 M) of RPA is mediated by a “DNA-binding core” (DBC) containing the four central OB-fold domains (70A, 70B, 70C, and 32D) with an occluded site size of 30 nucleotides and a 5′ to 3′ binding polarity from 70A to 32D (1, 2). Protein recruitment is mediated primarily by domains 70N and 32C and to some extent by the principal DNA-binding domains 70A and 70B (3).

RPA is believed to proceed through three different interaction modes upon binding ssDNA: an initial 8–10 nucleotide binding mode that includes domains 70A and 70B, a 12–23 nucleotide mode that proceeds to engage 70A–70C, and a final 28–30 nucleotide mode that encompasses all four DNA-binding domains (3). Collectively, these discrete interaction modes form a “DNA-binding trajectory” that RPA traverses as it progressively engages ssDNA and presumably modulate interaction with other DNA-processing factors. The intrinsic inter-domain flexibility within RPA-DBC (12, 13), however, has made structural characterization of these protein–DNA complexes particularly challenging. Integration of low-resolution spatial information from small-angle X-ray and neutron scattering (SAXS/SANS) with computational simulation and existing high-resolution structures (14–19) is a powerful approach for obtaining information on both the global architecture and disposition of individual domains in modular, multi-domain proteins (14). Successful application of this integrated approach to any system, though, depends heavily upon the ability to acquire high-quality scattering data, which necessitates a pure, homogeneous, and stable sample (16, 20). DNA-binding proteins such as RPA present a number of challenges for such sample production, due to their sensitivity to ionic strength and reducing environment, their potential to aggregate at high concentrations (in excess of 2–3 mg/mL), and their capacity to bind DNA in multiple states during attempts to form stoichiometric complexes.

Here we outline a general strategy for preparing protein–DNA systems for study by small-angle scattering, using RPA-DBC as a specific example. This strategy is organized into three stages: (1) screening stable buffer conditions, (2) assessing sample aggregation and stoichiometric complex formation by SEC-MALS, and (3) preparative purification of specific protein–DNA complexes for both SAXS and SANS.

2. Materials

2.1. Optimal Buffer Solubility Screening

2.1.1. Primary pH Solubility Screening

  1. Screening buffers (100 mM buffer, 50 mL each, refer to Fig. 1 for complete list).

  2. 60-mL syringes and 0.45-µm syringe filters.

  3. 50 mL sterile conical tubes.

  4. 200 µL RPA-DBC protein prepared at 1 mg/mL in 10 mM HEPES (pH 7.5), 100 mM NaCl, and 10 mM β-mercaptoethanol (βME) (21).

  5. 10.0 mM dC30 oligonucleotide stock in sterile water.

  6. 96-well sitting drop crystallography plates.

  7. Transparent packing tape.

  8. Tape applicator.

  9. Dissection microscope.

Fig. 1.

Fig. 1

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Sample scoring sheet for primary pH solubility screen.

2.1.2. Primary Ionic and Additive Solubility Screening

  1. Screening buffers (15 mL each, refer to Fig. 2 for complete list)

  2. 30-mL syringes and 0.45-µm syringe filters.

  3. 15 mL sterile conical tubes.

  4. RPA-DBC protein (Subheading 2.1.1).

  5. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

  6. 96-well sitting drop crystallography plates.

  7. Transparent packing tape.

  8. Tape applicator.

  9. Dissection microscope.

Fig. 2.

Fig. 2

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Organization of 96-well plate for primary pH solubility screen.

2.1.3. Secondary Solubility Screening

  1. Screening buffer (Subheading 3.3, refer to Figs. 1 and 2).

  2. 40 mL RPA-DBC protein prepared at 0.5 mg/mL (Subheading 2.1.1).

  3. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

  4. Amicon Ultra centrifugal concentrators (15 mL, 30 kDa MWCO, Millipore).

  5. 2× SDS Loading Buffer: 100 mM Tris–HCl, pH 6.8, 4% (w/v) SDS (electrophoresis grade), 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200 mM βME (added fresh).

2.2 Assessing Sample Monodispersity by SEC-MALS

2.2.1. Preparing, Equilibrating, and Calibrating the SEC-MALS System

  1. In-line system for gel filtration chromatography and measurement of UV absorbance, static light scattering, and differential refractive index (see Note 18).

  2. Superdex 200 PC 3.2/30 column (GE Healthcare).

  3. Screening buffer (Subheading 3.3), supplemented with 0.05% sodium azide.

  4. Bovine albumin, monomer, lyophilized (Sigma).

  5. 0.22-µm centrifugal spin filters.

  6. 96-well round-bottom plate (0.5 mL) (Agilent Technologies).

2.2.2. Sample Preparation and SEC-MALS Acquisition for Concentration Series

  1. Amicon Ultra centrifugal concentrators (15 mL, 30 kDa MWCO, Millipore).

  2. 20 mL RPA-DBC protein prepared at 0.5 mg/mL (Subheading 2.1.1).

  3. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

2.2.3. Sample Preparation and SEC-MALS Acquisition for Time Series

  1. Amicon Ultra centrifugal concentrators (15 mL, 30 kDa MWCO, Millipore).

  2. 30 mL RPA-DBC protein prepared at 0.5 mg/mL (Subheading 2.1.1).

  3. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

2.3. Preparative Sample Purification for SAXS and SANS

2.3.1. Preparative Purification for SAXS

  1. FPLC purification system and accessories.

  2. Gel filtration buffer (Subheading 3.3).

  3. Superdex 200 HR 10/30 column (GE Healthcare).

  4. Amicon Ultra centrifugal concentrators (15 mL, 30 kDa MWCO, Millipore).

  5. Amicon Ultra centrifugal concentrators (4 mL, 10 kDa MWCO, Millipore).

  6. 0.22-µm centrifugal spin filters.

  7. 15 mL RPA-DBC protein prepared at 0.5 mg/mL (Subheading 2.1.1).

  8. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

2.3.2. Preparative Purification for SANS

  1. FPLC purification system and accessories.

  2. Hydrogenated gel filtration buffer (Subheading 3.3).

  3. Deuterated gel filtration buffer (Subheading 3.3).

  4. Superdex 200 HR 10/30 column (GE Healthcare).

  5. Amicon Ultra centrifugal concentrators (15 mL, 30 kDa MWCO, Millipore).

  6. Amicon Ultra centrifugal concentrators (4 mL, 10 kDa MWCO, Millipore).

  7. 0.22-µm centrifugal spin filters.

  8. 15 mL RPA-DBC protein prepared at 0.5 mg/mL (Subheading 2.1.1).

  9. 10.0 mM dC30 oligonucleotide (Subheading 2.1.1).

3. Methods

This section describes three aspects of optimizing sample homogeneity and monodispersity in preparing a system for study by SAXS or SANS. The first part outlines a screening procedure for selecting and testing buffer conditions to ensure maximum sample solubility and stability under the acquisition environments of each scattering technique. The second part details the application of size-exclusion chromatography in-line with multi-angle light scattering (SEC-MALS) to provide further refinement of buffer conditions by assessing the potential for sample aggregation. The final section describes a scheme for preparative purification of protein–DNA complexes under refined buffer conditions prior to SAXS or SANS data acquisition.

3.1. Optimal Buffer Solubility Screening

The primary buffer screening assay is based upon that described by Howe (22) and implements a 96-well crystal screening format using a minimal amount of protein. Primary screening is performed in two separate, progressive rounds to assay first for optimal pH, then ionic strength and common additives. Successful buffer conditions revealed by primary screening are then tested on a larger scale at concentrations, temperatures, and timescales relevant to SAXS and SANS, respectively. Screening should be performed in parallel on both the protein alone and in complex with DNA, as we have found that binding of ssDNA substrates improves the stability of RPA. Consecutive rounds of primary and secondary screening will each require 2–3 days to complete.

3.1.1. Primary pH Solubility Screening

  1. Prepare 50 mL each of 100 mM buffer stocks (Fig. 1), adjust to target pH as needed, and filter at 0.45 µm into sterile conical tubes (see Notes 1 and 2).

  2. Prepare 200 µL stocks of fresh RPA-DBC and RPA-DBC/dC30 complexes at concentrations of 1 and 2 mg/mL in a buffer containing 10 mM HEPES (pH 7.5), 100 mM NaCl, and 10 mM βME. RPA-DBC/dC30 stock can be prepared by mixing RPA-DBC with 1.1-fold excess dC30 oligonucleotide (see Notes 3–7).

  3. Collect two fresh 96-well plates and clear all dust and fibers with pressurized air. Mark and divide the plates into four sections by rows (i.e., 2 rows per section, Fig. 2). Fill each reservoir of the first row with 100 µL of each test buffer solution and repeat for the remaining rows. For the reservoir designated as “Control,” provide the starting buffer for RPA-DBC (see step 2).

  4. Distribute 1 µL RPA-DBC stock at 1 mg/mL into the wells of the first two rows (A, B) and repeat for RPA-DBC at 2 mg/mL for rows C and D (Fig. 2). Repeat for RPA-DBC/dC30 stock at 1 and 2 mg/mL for the rows that remain. This should provide replicate tests of each buffer at two sample concentrations.

  5. Draw up 1 µL buffer from the reservoir, add to the protein drops, and mix carefully (see Note 8).

  6. Apply transparent packing tape over the surface of each plate and seal thoroughly to prevent evaporation.

  7. Incubate plates at 4°C (control) and 25°C and visually assess each drop for precipitation with a dissection microscope (according to the metric provided in Fig. 1) for 15 min, 1 h, 3 h, and overnight incubation (see Note 9).

  8. Once the assay has been completed, a target pH should be selected based upon conditions at 25°C that reflect scores in the range of 0–1 (faint or no precipitation) over the course of 3 h (see Note 10) and show an improvement upon the starting buffer (control reservoir). If the screen offers no improvement relative to the starting buffer control, screening may proceed to the next stage with the original buffer and pH (here, HEPES at pH 7.5). Should the screening fail to return any satisfactory conditions, the screen may need to be repeated utilizing a different starting buffer for the protein (i.e., higher ionic strength, etc.).

3.1.2. Primary Ionic and Additive Solubility Screening

  1. Prepare 15 mL of a buffer containing 100 mM of the buffer stock selected from the pH screen combined with 100, 200, or 500 mM NaCl or KCl (Fig. 3). Prepare a parallel set of identical buffers containing 100 mM buffer, 100 mM NaCl, and 2, 5, or 10% glycerol. Adjust to target pH as needed and filter at 0.45 µm into sterile conical tubes. This should provide a total of twelve buffers (Fig. 3, see Notes 2 and 11).

  2. Collect two fresh 96-well plates and clear all dust and fibers with pressurized air. Mark and divide the plates into four sections by rows (i.e., 2 rows per section, Fig. 3). Fill each reservoir of the first row with 100 µL of each test buffer solution and repeat for the remaining rows. For the reservoir designated as “Control,” provide the starting buffer for RPA-DBC (see Subheading 3.1.1).

  3. Aliquot 1 µL protein into each well as described in Subheading 3.1.1, carefully mix each well with 1 µL reservoir buffer, and seal the plates with transparent packing tape.

  4. Incubate plates at 4°C (control) and 25°C and visually assess each drop for precipitation with a dissection microscope (according to the metric provided in Fig. 1) for 15 min, 1 h, 3 h, and overnight incubation (see Note 9).

  5. Once the assay has been completed, a target salt and glycerol concentration should be selected based upon conditions at 25°C that reflect scores in the range of 0–1 (faint or no precipitation) over the course of 3 h (see Note 10) and show an improvement upon the starting buffer (control reservoir). If the screen offers no improvement relative to the starting buffer control, screening may proceed to the next stage with the original buffer conditions. Should the screening fail to return any satisfactory conditions, the screen may need to be refined and repeated with a different set of additives (Table 1, see Note 11).

Fig. 3.

Fig. 3

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Sample scoring sheet for primary ionic and additive solubility screen.

Table 1.

Additional additives and counterions for primary screening

MgCl2 (NH4)SCN NaOAcetate
MgSO4 NaSCN (NH4)OAcetate
CaCl2 KSCN NaCitrate
MnCl2 NaSO4 NaTartrate
LiCl (NH4)SO4 NaFormate
Imidazole Arginine Glutamate
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3.1.3. Secondary Solubility Screening

Once a candidate buffer has been chosen from the primary screens, the buffer must be tested under concentrations and timescales relevant to SAXS (1–10 mg/mL, 15–60 min) and SANS (1–5 mg/mL, 1–12 h). For SAXS, data acquisition is rapid (seconds), and the sample lifetime need only encompass the time required to transport the freshly prepared sample from an on-site preparative laboratory to initiating the experiment at the beamline. For SANS, where the neutron beam flux is much lower and thus acquisition times much longer, the sample lifetime must extend through the hours needed for data collection. In addition to this, collection of a SANS contrast variation series (see Note 12) also requires exchanging the sample into a deuterated version of the buffer, which may exhibit pronounced differences in solubility. Below is a procedure for testing sample lifetime under these different conditions, where the sample is first transferred into the target buffer, then monitored for 24 h across a concentration series (1, 2, 5, 10 mg/mL) at room temperature and 4°C.

  1. Prepare 4 L of the target buffer, adjust to the target pH, and prechill to 4°C (see Note 13).

  2. Dialyze 40 mL of freshly prepared RPA-DBC at 0.5 mg/mL into the target buffer overnight at 4°C with gentle stirring.

  3. Prerinse a centrifugal concentrator unit (15 mL, 30 kDa MWCO, Millipore) with Milli-Q water (filtered at 18.2 Ω) for 10 min at 3,700 × g in a refrigerated (4°C) tabletop centrifuge. Concentrate the dialyzed protein to ~1.6 mL (or restore the concentrate to this volume with flow-through buffer if the volume is less) and measure the concentration by absorbance at 280 nm with a UV–Vis spectrophotometer (see Note 14).

  4. Divide the concentrate in half and add 1.1-fold molar excess dC30 oligonucleotide to one half of the protein stock to prepare complexes of RPA-DBC and ssDNA. Mix well and incubate for 15–20 min (see Note 15).

  5. Prepare two 100-µL aliquots for each sample (RPA-DBC and RPA-DBC/dC30) at the following concentrations: 1, 2, 5, and 10 mg/mL. This should result in 4 concentration series (16 aliquots total, Fig. 4).

  6. Collect an initial concentration reading from each aliquot using a Nanodrop (2 µL material required per reading, see Note 16). Incubate one concentration series for each sample (RPA-DBC and RPA-DBC/dC30) at 4°C and the remaining series at room temperature.

  7. Observe the samples after 15 min for any visible clouding of the solutions. At 30 min, 1 h, 3 h, 6 h, and 24 h (overnight) (Fig. 4) examine the samples for any visible precipitation. Spin the samples 5 min at 16,000 × g in a refrigerated Eppendorf tabletop centrifuge. Note if any precipitated material has accumulated at the bottom of each tube, then collect concentration measurements by Nanodrop. Collect 10 µL of each sample and combine with 10 µL 2× SDS-PAGE loading buffer for evaluation by SDS-PAGE.

  8. At the completion of the assay, run an SDS-PAGE gel of all samples to ensure an absence of degradation during the incubation periods. Assess fluctuations in sample concentration to select a target concentration and time frame over which sample stability can be assured. As mentioned previously, a minimum sample concentration of 1–2 mg/mL that can maintain stability for 15–60 min (SAXS) or 1–12 h (SANS) is preferred. If the sample fails to do well under scale-up conditions, another round of primary screening can be employed to determine additional additives that may improve stability.

Fig. 4.

Fig. 4

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Sample scoring sheet for secondary solubility screen.

3.2. Assessing Sample Monodispersity by SEC-MALS

Once buffer conditions have been established via the protocol outlined in the previous section, it is critical to confirm that the sample is not invisibly aggregating under these conditions. Size-exclusion chromatography and multi-angle light scattering (SEC-MALS) are arguably the most effective means to confirm sample homogeneity and monodispersity prior to investigation by SAXS or SANS. As in the previous section, monodispersity is assayed for select sample concentrations and incubation periods, dependent upon the results of the secondary screening (Subheading 3.1.3). In the interest of completeness, we describe assessment of the full concentration series (1, 2, 5, 10 mg/mL) and time course (30 min, 1 h, 3 h, 6 h, 24 h) (see Note 17).

3.2.1. Preparing, Equilibrating, and Calibrating the SEC-MALS System

  1. Exchange the pump and FPLC system into a solution of 0.05% sodium azide in Milli-Q water filtered fresh at 0.2 µm (see Notes 18, 19). With the flow rate set to 0.01 mL/min, make a drop-to-drop connection to the inlet of the 2.4 mL Superdex 200 PC 3.2/30 column and immediately connect the outlet to the UV detector. Gradually increase the flow rate to 0.04 mL/min in 0.01 mL/min increments (allow 4–5 min equilibration for each new flow rate). Allow the column and system to equilibrate for 1.5 column volumes (CV), approximately 90 min, with purging of the reference cell for the refractive index (RI) unit.

  2. Disconnect the column under flow and close off the ends. Exchange the pump and FPLC system into the target test buffer, freshly filtered at 0.2 µm. As before, make a drop-to-drop connection to the inlet of the 2.4 mL Superdex 200 PC 3.2/30 column at 0.01 mL/min and immediately connect the outlet to the UV detector. Gradually increase the flow rate to 0.04 mL/min in 0.01 mL/min increments. Allow the column and system to equilibrate at least 3–4 CV, preferably overnight, to ensure a stable baseline for the RI detector. Do not stop the flow of buffer until all experiments are complete, as the equilibration period will have to be repeated to restabilize the RI baseline.

  3. Once the RI baseline has stabilized, prepare a fresh 500-µL solution of monomeric BSA at 10 mg/mL in the target buffer. Filter 50 µL of the stock BSA solution using a 0.22-µm centrifugal spin filter in a refrigerated Eppendorf tabletop centrifuge (see Note 20). Load 45 µL of the filtered BSA solution into a round-bottom 96-well plate and insert into the auto-injection module of the system. Take care not to disturb the column or lines connecting the RI module.

  4. Prepare a ChemStation method to inject a 40 µL sample and then run for 98 min at the target flow rate and pressure limit for the Superdex 200 PC 3.2/30 column (here, 0.04 mL/min and 15 bar). This includes 3 min for completing the auto-injection, 90 min of elution time, and 5 min for a COMET cleaning of the light scattering flow cell (see Note 21). Prepare an equivalent recording session in Astra V to record for 90 min with the 5-min COMET cleaning enabled (see Note 22).

  5. Implement the Astra recording session, which should pause and await sample auto-injection from the ChemStation software (see Note 22). Initiate the ChemStation method, ensure that there are no issues with the sample auto-injection, and confirm that Astra begins recording data.

  6. Once the run has finished, the system should continue to flow buffer through the column according to the rate and pressure limit set prior to starting the run. Save the Astra session (the data is not automatically saved by the program). Apply baseline corrections and define a peak region about the primary monomeric BSA peak, taking care to avoid any contributions from stray higher-order species. Apply peak normalization, alignment, and band broadening against the UV, light scattering, and refractive index traces as described in the Astra V user’s guide. Confirm that the average molecular weight (Mr) of the monomeric peak is ~66 kDa and that the polydispersity is 1.000 (see Note 23).

  7. Save the calibrated session as a template and start all new experiments from the BSA template.

3.2.2. Sample Preparation and SEC-MALS Acquisition for Concentration Series

  1. Prepare 20 mL of freshly purified RPA-DBC at 0.5 mg/mL in the target buffer and concentrate to ~1 mL in a prerinsed centrifugal concentrator unit (15 mL, 30 kDa MWCO, Millipore) (see Note 14). Assess protein concentration by absorbance at 280 nm with a UV–Vis spectrophotometer. Divide the RPA-DBC stock in half and add 1.1-fold molar excess dC30 oligonucleotide to one half; mix well and incubate for 20–30 min (see Note 15).

  2. Prepare 100-µL aliquots of each sample at 1, 2, 5, and 10 mg/mL and store on ice at 4°C. Filter 50 µL of the first sample solution (RPA-DBC, 1 mg/mL) using a 0.22-µm centrifugal spin filter in a refrigerated Eppendorf tabletop centrifuge. Load 45 µL of the filtered sample solution into a round-bottom 96-well plate and insert into the auto-injection module of the system. Take care not to disturb the column or lines connecting the RI module.

  3. Initiate the Chemstation and Astra sessions as before (Subheading 3.2.1) to inject the sample and record the subsequent elution profile. Once the run has finished, save the Astra session, apply baseline corrections and define peak regions for all peaks present on the chromatogram. The normalization, alignment, and band broadening procedures need only be applied to the calibration run and should already be encoded in the experiment template used for the subsequent runs on RPA-DBC.

  4. In the results section of the Astra session, examine the average Mr and polydispersity (Is the Mr close to the expected value? Is the polydispersity close to 1.000 with a relatively low percent error?). In the EASI graph section of the program, inspect the light scattering chromatogram versus that for UV absorbance. (Is the primary peak symmetric? Does the peak tilt or are there any shoulders present? Does the sample elute in the void volume?) Using the EASI graph feature, plot the molar mass over the light scattering chromatogram and determine the consistency of the molar mass across the span of the eluted peak. (Is the molar mass estimation flat across the width of the peak or is it sloped?) Compare the molar mass distribution with that seen for the BSA calibration run. Aggregation can be manifest as an upturn in molar mass toward the early eluting edge of the peak or the presence of a peak in the corresponding void volume of the column (see Note 24).

  5. Repeat the run procedure for the remainder of the concentration series and overlay the resulting light scattering chromatograms to determine if there is a concentration-dependent appearance of aggregation at the void volume and/or upturn in the molar mass.

3.2.3. Sample Preparation and SEC-MALS Acquisition for Time Series

  1. Prepare 30 mL of freshly purified RPA-DBC at 0.5 mg/mL in the target buffer and concentrate to ~1.5 mL in a prerinsed centrifugal concentrator unit (15 mL, 30 kDa MWCO, Millipore). Assess protein concentration by absorbance at 280 nm with a UV–Vis spectrophotometer. Divide the RPA-DBC stock in half and add 1.1-fold molar excess dC30 oligonucleotide to one half; mix well and incubate for 20–30 min (see Note 15).

  2. Prepare 6 100-µL aliquots at the target concentration determined previously (Subheading 3.2.2). These will correspond to incubation periods of 30 min, 1 h, 3 h, 6 h, and 24 h at room temperature. Because each SEC-MALS run requires 1.5–2 h, the incubation periods must be staggered accordingly.

  3. Sample filtration and loading are performed as described (Subheading 3.2.2), as well as data analysis, once each run has been saved. Once all runs have been completed, an overlay of light scattering chromatograms should reveal if there is a time-dependent appearance of aggregation at the void volume and/or upturn in the molar mass.

3.3. Preparative Sample Purification for SAXS and SANS

As a final precaution against aggregation and to promote homogeneous formation of protein–DNA complexes, all scattering samples should be passed over a final gel filtration column prior to data acquisition. Because of the reduced sample requirements for SAXS, the elution profile can be finely parsed and sampled to select for the most homogeneous portion of the eluting peak (see Note 25). The increased sample needs for SANS, though, may require combining a broader section of the eluting peak; thus minimizing all forms of aggregation during buffer optimization is particularly critical (see Note 26). In this last section, we describe basic protocols for preparative gel filtration of samples for SAXS and SANS, as well as preparation of the resulting gel filtration fractions for data acquisition.

3.3.1. Preparative Purification for SAXS

Sample loading and elution from the gel filtration column are implemented using an automated FPLC method. This protocol is designed to generate samples across the base of the main eluting peak, with concentration series based upon each fraction (1×, 2×, and 4×) (see Note 27).

  1. Equilibrate the Superdex 200 HR 10/30 column with 1.5 CV filtered Milli-Q water (36 mL) and at least 3 CV filtered hydrogenated sample buffer (72 mL). The column should be connected to the system under flow (0.1 mL/min), with gradual increase of the flow rate every 5 min by 0.1 mL/min until the target flow rate of 0.3 mL/min is reached (see Note 28).

  2. Prerinse one centrifugal concentrator unit (15 mL, 30 kDa MWCO, Millipore) with Milli-Q water for 10 min at 3,700 × g in a refrigerated (4°C) tabletop centrifuge. Concentrate 15 mL freshly prepared RPA-DBC protein at 0.5 mg/mL to ~600 µL (see Note 14).

  3. Assess protein concentration by absorbance at 280 nm with a UV–Vis spectrophotometer. Divide the RPA-DBC stock in half and add 1.1-fold molar excess dC30 oligonucleotide to one half; mix well and incubate for 20–30 min (see Note 15).

  4. Filter the first DNA-free sample (~300 µL, ~12 mg/mL) using a 0.22-µm centrifugal spin filter in a refrigerated Eppendorf tabletop centrifuge and load onto the Superdex 200 HR 10/30 column at 0.3 mL/min, collecting 290 µL fractions in Eppendorf tubes for a 1.5 CV (36 mL) elution period (see Note 29). Store the fractions at 4°C as they come off the column.

  5. Repeat the purification with the RPA/dC30 sample.

  6. Using a Nanodrop, assay absorbance for each fraction at 260 and 280 nm to determine protein concentration and to assess DNA-binding for RPA/dC30 according to the A260/A280 ratio (see Note 30)

  7. Select fractions that are 1 mg/mL or greater and reserve 25 µL each for data acquisition (see Note 31). Select an additional 3 fractions from the void volume (typically 7–8 mL into the elution, which should exhibit a flat UV baseline) to serve as samples for buffer background subtraction.

  8. Prerinse one centrifugal concentrator unit per fraction (4 mL, 10 kDa MWCO, Millipore) with Milli-Q water for 10 min at 3,700 × g in a refrigerated (4°C) tabletop centrifuge. Rinse the concentrators two additional times with gel filtration buffer. Concentrate the remainder of each fraction (~265 µL) to ~130 µL (2×), remove 25 µL for data acquisition, and continue concentration to ~60 µL (4×) (see Note 32).

  9. The original fractions (1×) and concentrated fractions (2× and 4×) should be kept at 4°C until transfer to a 96-well plate for SAXS data acquisition (no less than 24 h after gel filtration).

3.3.2. Preparative Purification for SANS

Sample loading and elution from the gel filtration column are implemented using an automated FPLC method. Sample preparation proceeds in three stages: (1) gel filtration exchange of RPA-DBC/dC30 into H2O- and D2O-based buffers, (2) preparation of concentrated stocks of RPA-DBC/dC30 from each purification, and (3) mixing samples for the contrast variation series.

  1. Equilibrate the Superdex 200 HR 10/30 column with 1.5 CV filtered Milli-Q water (36 mL) and at least 3 CV filtered hydrogenated sample buffer (72 mL). The column should be connected to the system under flow (0.1 mL/min), with gradual increase of the flow rate every 5 min by 0.1 mL/min until the target flow rate of 0.3 mL/min is reached (see Note 28).

  2. Prerinse one centrifugal concentrator unit (15 mL, 30 kDa MWCO, Millipore) with Milli-Q water for 10 min at 3,700 × g in a refrigerated (4°C) tabletop centrifuge. Concentrate 15 mL freshly prepared RPA-DBC protein at 0.5 mg/mL to ~600 µL (see Note 14).

  3. Assess protein concentration by absorbance at 280 nm with a UV–Vis spectrophotometer. Add 1.1-fold molar excess dC30 oligonucleotide to the entire stock, mix well, and incubate for 20–30 min. Divide the RPA-DBC/dC30 mixture in half.

  4. Filter the first RPA-DBC/dC30 sample (~300 µL, ~12 mg/mL) using a 0.22-µm centrifugal spin filter in a refrigerated Eppendorf tabletop centrifuge and load onto the Superdex 200 HR 10/30 column at 0.3 mL/min, collecting 500 µL fractions in Eppendorf tubes for a 1.5 CV (36 mL) elution period (see Note 29). Store the fractions at 4°C as they come off the column.

  5. Re-equilibrate the Superdex 200 HR 10/30 column with at least 3 CV filtered deuterated sample buffer (72 mL) and proceed with the purification of the remaining RPA/dC30 sample as in step 4.

  6. Using a Nanodrop, assay absorbance for each fraction at 260 and 280 nm to determine protein concentration and to assess DNA-binding for RPA/dC30 according to the A260/A280 ratio.

  7. A five-point contrast variation series (0, 10, 20, 80, and 100% D2O buffers) with 325-µL samples will require ~940 µL of H2O sample stock and ~680 µL D2O sample stock to generate all five samples. Starting from the fraction containing the peak maximum of each H2O and D2O elution profile, determine which fractions from each purification would be required to generate H2O and D2O at the target sample concentration (at least 1–2 mg/mL) at the given volumes. Select an additional 3 fractions from the void volume (typically 6–7 mL into the elution, which should exhibit a flat UV baseline) to prepare samples for buffer background subtraction.

  8. Prerinse two centrifugal concentrators (4 mL, 10 kDa MWCO, Millipore) with Milli-Q water for 10 min at 3,700 × g in a refrigerated (4°C) tabletop centrifuge. Rinse the concentrators an additional time with gel filtration buffer. Pool and concentrate the selected fractions from each purification to the target volumes for each stock (see Note 32).

  9. Transfer the concentrates to Eppendorf tubes and spin down any precipitation in a refrigerated Eppendorf tabletop centrifuge for 10 min at 16,000 × g. Assess concentration by absorbance at 260 and 280 nm.

  10. Prepare 10, 20, and 80% D2O mixtures of RPA-DBC/dC30 and equivalent buffer blanks by volume (325-µL per sample). Measure A260 and A280 a final time and store samples and the remaining 0 and 100% D2O stocks on ice at 4°C until acquisition. Transfer samples to clean quartz cells after spinning a final time for 10 min at 16,000 × g and allow samples to equilibrate at room temperature for 20–40 min to allow outgassing (see Note 33).

Acknowledgements

The authors would like to thank Dr. Robert Rambo at Lawrence Berkeley National Laboratory for sharing his extensive expertise in the use of SEC-MALS, as well as Dr. Kevin Weiss and Dr. William Heller at the Center for Molecular and Structural Biology at Oak Ridge National Laboratory for helpful discussions regarding SANS sample preparation strategies. This work was supported by the National Institutes of Health operating grants R01 GM65484 and P01 CA092584.

Footnotes

1

Selected buffers for pH testing are based upon those described by Jancarik and colleagues (23) and encompass a range of pH 5–9.

2

Buffer stocks which are not to be used immediately may be stored long term at −80°C.

3

In the absence of ssDNA substrate, RPA-DBC aggregates at concentrations in excess of 2.5 mg/mL. While this critical concentration is likely to vary from system to system, a starting concentration of at least 1–2 mg/mL is preferred, as this is the minimum concentration range recommended for scattering studies. Follow-up screening at higher concentrations can be pursued once stable conditions have been established for the minimum concentration.

4

The concentration of HEPES (10 mM) within the protein buffer is kept low relative to that described in the original purification procedure to ensure that the primary buffering capacity arises from the screening buffer (i.e., 50 mM test buffer versus 5 mM HEPES).

5

Components of the starting buffer will vary and may be subject to optimization as well, depending upon the system of interest. For various RPA constructs, starting buffers have included 50–100 mM NaCl and 2–10 mM BME to provide ionic stabilization to the protein’s basic OB-folds and full reduction of the cysteine resides coordinating the metal center of the zinc ribbon of the 70C domain.

6

βME should be added fresh to the buffer prior to use.

7

Protein stocks should be maintained on ice at 4°C.

8

Care is needed in mixing, as this can introduce air bubbles to the protein drops.

9

Precipitation may manifest in a number of ways, whether a fine clouding of the drop or the dramatic appearance of brown/black conglomerates. The metric provided in Fig. 1 reflects the assumption that the severity of the precipitation is reflected by the density of the precipitation within the drop (a few grains versus complete occupation of the drop). For more details, refer to Howe (22).

10

In our experience, the majority of precipitation occurs within the first 15–20 min after mixing; if a drop remains clear beyond this, it is likely to remain relatively clear through the remainder of the incubation period.

11

The choice of the salts NaCl and KCl and the additive glycerol for the second round of screening is based on past successes with various RPA constructs. A list of ionic agents and additives that may be tested in addition to these is provided in Table 1.

12

A SANS contrast variation series encompasses five to six samples of the target protein–DNA complex prepared in buffers containing different mixtures of H2O and D2O as solvent (typically 0, 10, 20, 30, 80, and 100% D2O). Differences in scattering contrast for protein and DNA under these different ratios of H2O and D2O allow their respective scattering contributions to be parsed from the global scattering of the complex (24). Because D2O has very different physical properties compared to H2O (including differential hydrogen bonding and acid/base behavior), protein solubility may be negatively affected when substituting D2O as the primary buffer solvent.

13

Due to the expense of D2O, we recommend running all secondary solubility testing on hydrogenated buffers first, before proceeding with testing of deuterated buffers. Typically, SANS is contemplated for a protein–DNA complex once successful data acquisition and analysis have been achieved by SAXS; as such, deuterated buffers may not be required for some time. When this stage of the analysis is reached, we recommend embarking on a small-scale version of the secondary testing described here, employing a 100–300 mL dialysis buffer with protein volume scaled accordingly.

14

Concentrators are usually spun in 10–15 min increments with careful mixing upon each addition of protein to prevent buildup and aggregation of protein at the base of the concentrator.

15

In the interest of preserving protein stability prior to exposing a sample to long-term incubation at room temperature, we have historically prepared protein–DNA mixtures on ice. If protein stability is assured, incubation at room temperature is preferred in order to ensure complete equilibration of protein–DNA binding.

16

In our experience, absorbance measurements by UV–Vis spectrophotometer return more accurate assessments of concentration on more dilute protein solutions compared to Nanodrop. Absorbance measurement by Nanodrop, however, requires the expenditure of less protein. In light of this, we describe measuring the initial concentration of the original stock solutions by UV–Vis spectrophotometery, then following subsequent changes in concentration for each series by Nanodrop.

17

As mentioned before for the secondary solubility screening assay, testing of deuterated buffers should be considered only after hydrogenated buffers have been thoroughly optimized and at least one round of study by SAXS has been successfully completed. A 36-h SEC-MALS run using a 2.4 mL Superdex 200 PC 3.2/30 column would be expected to consume ~120 mL of buffer.

18

Our SEC-MALS system includes in-line detectors for ultraviolet absorbance at 280 nm (Agilent 1100 series, Agilent Technologies), static light scattering (DAWN HELEOS 8+, Wyatt Technology), and differential refractive index (Agilent 1200 series, Agilent Technologies). The system is equipped with an automated injection system (Agilent 1100 series, Agilent Technologies). Operation of the FPLC system is managed by Agilent ChemStation software, while data recording and analysis are carried out by Astra V (version 5.3.4.18).

19

It is critical that all solutions introduced to the FPLC system are freshly filtered and supplemented with 0.05% sodium azide to prevent any particulate matter from potentially clogging the light scattering flow cell and to inhibit microbial growth and contamination of the system.

20

Allow approximately 15 and 30 min, respectively, for the light scattering laser and UV lamp to warm up prior to final preparation of the first sample.

21

Since all modifications to ChemStation methods are implemented in real time, we recommend preparing and uploading the ChemStation method prior to connecting the column to the instrument for equilibration to prevent unexpected changes in flow rate or maximum pressure allowances.

22

Our system has been setup to initiate recording automatically in Astra once sample auto-injection is complete.

23

If there is a discrepancy in the measured and predicted molecular weights, confirm that the definitions of the peak boundaries exclude any trace aggregation that may elute as a shoulder to the main BSA peak. (We observe slight dimer formation, even with the monomeric standard). If the discrepancy still remains, Astra’s default value for the buffer refractive index may not be accurate for the test buffer (the default parameters are based upon phosphate buffered saline). This value can be measured with a refractometer and adjusted under the solvent settings of the experiment.

24

In the case of protein–DNA complexes, signs of aggregation by SEC-MALS may in fact highlight the presence of non-stoichiometric binding (two or more DNA molecules per protein molecule or vice versa). The addition of 1.1-fold molar excess oligonucleotide for RPA is intended to ensure full saturation of all binding sites without providing opportunity for multiple DNA molecules to associate with a single RPA molecule. For example, we find that the addition of threefold molar excess of DNA produces an early eluting shoulder in addition to the primary peak of the complex. If a non-stoichiometric interaction is suspected, we recommend varying the molar ratio of DNA to protein when forming the complex or adjusting the salt concentration of the buffer.

25

Each SAXS sample requires ~20 µL at the target concentration (minimum of 1 mg/mL).

26

A five-point SANS contrast variation series will require five 325-µL samples (~1.6 mL) at a minimum of 1–2 mg/mL. Sample volume assumes the use of 1 mm quartz banjo cells.

27

Collecting SAXS data on a concentration series of the sample allows for assessment of inter-particle interference while maximizing signal sensitivity (17). In our case, a concentration series is preferred to a dilution series, as irreversible aggregation that could be induced at higher concentrations is not propagated to more dilute samples.

28

All gel filtration columns will shed a certain amount of their matrix upon first application of liquid flow pressure and then will stabilize. In order to minimize introduction of this matrix material into the eluting sample, it is recommended that flow be maintained after column equilibration and between individual runs. If the column must be left for an extended period of time, simply reduce the flow rate to 0.05 mL/min and ensure that there is sufficient buffer in the pump reservoir.

29

The concentration of the sample will be diluted after passage through the gel filtration column. If the dilution factor of the column is known for a particular sample volume (a series of calibration runs can establish this), then the loading concentration can be adjusted to guarantee a final concentration of 1–2 mg/mL at the maximum point of the eluting peak.

30

We have also found that confirming concentration by Bradford assay is particularly helpful for protein–DNA complexes, as defining an accurate extinction coefficient for the mixed species can be difficult. If sample material is limited prior to data acquisition, the assay can be performed once the experiment is completed.

31

In our case, the peak for RPA-DBC without ssDNA is contained within three 290-µL fractions, each greater than 1.5 mg/mL.

32

The speed of concentration will depend upon the protein. We recommend spinning in 5 min increments with mixing in between to reduce the risk of overshooting the target concentrations and potentially inducing aggregation.

33

As a cold aqueous sample comes to room temperature, excess dissolved gas will come out of solution and form bubbles on the face of the quartz cells that can interfere with sample scattering.

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