Microfluidic Modulation Spectroscopy and Circular Dichroism Spectroscopy for In-Situ Detection of Structural Changes for Protein Structure Analysis

Microfluidic modulation spectroscopy (MMS) is a highly sensitive and intuitive instrument for secondary structure analysis.¹ MMS provides a more powerful alternative to circular dichroism (CD) spectroscopy, an analytic approach previously utilized to identify secondary structure.

A biological protein sample was utilized in this study to explore the performance of MMS compared to CD for secondary structure analysis. The biological function of the protein under investigation (protein X) varies alongside changes in concentration.

When diluted to 1 mg/mL, protein X behaves similarly to a monomer. However, it oligomerizes and works cooperatively at elevated concentrations of approximately 80 mg/mL. Protein X is additionally rich in β-sheets and has an unordered tail, which supposedly plays a key role in reorganization along the oligomerization mechanism.

This study investigated different concentrations of protein X, spanning 0.8 to 80 mg/mL, using both MMS and CD spectroscopy. The secondary structural analysis produced by these approaches was later compared.

MMS has previously been shown to be the most sensitive secondary structure method for detecting discreet structural changes, as little as 0.76 %.¹ Signals from different secondary structural motifs are well-defined over the probed spectral range and appear on an identical order of magnitude.

CD spectroscopy comparatively offers a dependable structural characterization of helix-rich proteins. However, it is less effective at calculating other structural content, including β-sheets, unordered structures, and turn structures.

This is due to CD signals from various structural motifs overlapping and having both positive and negative parts, which can ultimately cancel each other out. This makes deconvolution challenging due to destructive interference. The α-helical signal is significantly more powerful than those from different structural motifs, further contorting the structural deconvolution.

Figure 1 compares MMS and CD spectra for a helical protein, bovine serum albumin, and a β-sheet-rich protein, immunoglobulin G1.

Figure 1. MMS (left) and CD (right) spectra of BSA (dashed lines) and IgG1 (solid lines), highlighting the overlapping features and differing intensities for alpha-helix and beta-sheet structures in CD and compared to the more well-resolved peaks in MMS. This figure was adapted from Kendrick et al.¹ Image Credit: RedShiftBio

Methods

A concentration series of protein X was formulated in phosphate-buffered saline (PBS), producing concentrations ranging from 0.8 to 80 mg/mL. Samples were quantified and examined via a first-generation RedShiftBio MMS production system fitted with sweep scan capability (RedShiftBio, Boxborough, MA, US).

A chemically matching buffer was then loaded pairwise alongside the different samples onto a 96-well plate for background subtraction. The tool was subsequently run on a modulation frequency of 1 Hz alongside a microfluidic transmission cell with a 23.5 µm optical pathlength.

The differential absorbance spectra of the sample against its buffer reference were assessed across the amide I band (1,714–1590 cm^-1). Repeat assessments were gathered thrice and averaged, and data was examined with the delta analysis software for each spectrum.

Certain samples from the dilution series were additionally assessed via a Chirascan CS/PCS CD spectrometer utilizing a 0.01 mm quartz cuvette, specifically built for a wider concentration range, as outlined in Ioannou et al.²

Three repeat assessments were captured between 200 and 360 nm, each comprising 10 co-adds. CD data was quantified as mean residue ellipticity and integrated with the CDpro routine for secondary structure analysis.³

The two approaches were qualified by conducting a secondary structure analysis of the lysozyme sample, a principally α-helical protein. MMS and CD spectra of 1 mg/mL hen egg white lysozyme in water were gathered and assessed.

Results and Analysis

Method Qualification: MMS and CD Qualification Using Lysozyme

Before inspecting the oligomerization of protein X, 1 mg/mL lysozyme dissolved in water was utilized as a simpler and well-characterized example to validate the MSS and CD approaches utilized for structural characterization.

Figure 2 depicts higher-order structure (HOS) analysis via MMS and CD; crystallographic data from the protein database file 1DPX4 were used as a control.

Figure 2. Secondary Structure Analysis based on MMS and CD measurements of lysozyme at 1 mg/mL in water, compared to crystallographic data (pdb identifier: 1DPX).⁴ Image Credit: RedShiftBio

MMS and CD each distinguish around 45 % α-helix, 25 % turn structure, 15 % β-sheet, and 15 % unordered structure. This principally supports the lysozyme’s displayed crystal structure.

The two approaches marginally undercalculate turn structure at around 10 % and overcalculate β-sheet content, making lysozyme seem slightly better structured in solution than in its crystal structure.

MMS and CD analysis are in sound agreement with one another, emphasizing the applicability of the two approaches for analyzing simple, helix-rich proteins, including lower concentrations of lysozyme (1 mg/mL) when dissolved in water.

These approaches will be used to explore the concentration-dependent structure-function relationship of protein X powered by oligomerization to examine the suitability of MMS and CD for a more complex and biologically relevant system.

The experimental challenge of this research is that protein X is β-rich, the buffer background is PBS rather than water, and the concentration spectrum required for coverage is extensive (0.8–80 mg/mL).

Case Study: MMS and CD on the Oligomerization Mechanism of Protein X

MSS and CD spectra of protein X in PBS at different concentrations between 0.8 and 8 mg/mL were captured.

The general MMS spectral variations between elevated concentration samples and the 0.8 mg/mL sample were quantified via weighted spectral difference and are demonstrated in Figure 3. The most significant change surfaces between 1 and 20 mg/mL. Smaller variations are meanwhile visible at concentrations exceeding 20 mg/mL.

The Figure 3 inset resolves the associated spectral variations across the amide I band, showing that differences occur principally in the β-sheet and unordered range. The β-sheet band is depicted to grow as a function of concentration as the unordered band decreases.

Figure 3. The MMS Weighted Spectral Difference (WSD) across the measured concentration range between 0.8 and 80 mg/mL. Inset: Difference spectra with 0.8 mg/mL as the control spectrum, the background is color-coded according to the typical spectral ranges for the individual structural motifs. Image Credit: RedShiftBio

A Gaussian deconvolution model and a CDpro fitting routine were applied to the MMS and CD spectra to calculate the HOS of protein X at different concentrations. The results are displayed in Figure 4.

MMS analysis can predict around 55 % β-sheet, 30 % turn, 10 % α-helix, and 5 % unordered structure. Boosting concentration additionally boosts the β-sheet fraction by 2 % and reduces the unordered content by 2.9 %.

Following the proposed mechanism, the unordered tail of protein X underpins oligomerization by interacting with neighboring protein X monomers, becoming increasingly structured.

CD-based analysis inversely does not identify transient structural variation with increasing concentration in protein X. Significant structural composition variations are estimated (-40 % β-sheet fraction, +40 % helical content, and non-monotonic variations in turn and unordered content).

Such results suggest that CD analysis was not as good as lysozyme at estimating the protein’s structure, failing to identify crowding-induced structural variations.

Figure 4. HOS analysis based on MMS (top) and CD (bottom) spectra of protein X in PBS at different concentrations between 1.3 and 80 mg/mL. The MMS and CD spectra are shown each in the corresponding insets in the top right corners. The MMS spectra are presented as the inverted and baselined 2^nd derivative spectra, the CD spectra are shown as mean residue ellipticity. Image Credit: RedShiftBio

Conclusions

MMS has proven an acceptable approach for characterizing proteins with different structural profiles over various concentrations. MMS spectra can meanwhile be used to find discrete structural variations in proteins, the estimations of which support the formerly suggested oligomerization mechanism of protein X.

CD spectroscopy demonstrated an ability to deliver satisfactory outcomes from a helix-rich protein in water at lower concentrations. However, for protein X oligomerization, CD spectroscopy has difficulty delivering realistic HOS estimations for β-sheet-rich proteins.

References and Further Reading

1. Kendrick, B. S., et al. (2020) Determining Spectroscopic Quantitation Limits for Misfolded Structures. J Pharm Sci, 109, pp.933–936.
2. Ioannou, J. C., et al. (2015) Characterising the secondary structure changes occurring in high-density systems of BLG dissolved in aqueous pH 3 buffer. Food Hydrocoll, 46, pp.216–225.
3. Sreerama, N., et al. (2000) Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem, 287, pp.252–260.
4. Weiss, M. S., et al. (2000) Biological Crystallography Crystallization, structure solution and refinement of hen egg-white lysozyme at pH 8.0 in the presence of MPD. Acta Crytallographica, D56, pp.952–958.

About RedShiftBio

RedShiftBio is redefining the possibilities for analyzing protein structure and concentration.

RedShiftBio has developed a proprietary life sciences platform combining our Microfluidic Modulation Spectroscopy (MMS) and expertise in high-powered quantum cascade lasers that provide ultra-sensitive and ultra-precise measurements of molecular structure. These structural changes affect critical quality attributes governing the safety, efficacy, and stability of biomolecules and their raw materials. This combination of technologies is available to researchers in our fully-automated Aurora and Apollo systems and is backed by a global network of sales, applications, service, and support teams to address all market needs.

Alongside our commitment to further innovation in the formulations and development space, RedShiftBio also supports biopharmaceutical manufacturing with HaLCon, our bioprocess analytics platform, purpose-built to measure protein titer at time of need.

Led by an experienced management team with a proven track record of success in both large instrumentation companies and commercializing disruptive technologies, RedShiftBio is here to support your research, development, and manufacturing goals. Our instruments can be found in the majority of the leading biopharmaceutical companies and CDMOs in the world. We also run product demonstrations and process samples in the StructIR Lab, located in our Boxborough, MA headquarters, as well as at partner sites including the Wood Centre in Oxford, UK, Spectralys/UCB in Brussels, Belgium, and at Sciex laboratories in Redwood Shores, CA.

RedShiftBio is backed by Waters Corporation, Illumina Ventures, Technology Venture Partners, and one undisclosed leading life science company.

Sponsored Content Policy: AZoLifeSciences publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of AZoLifeSciences which is to educate and inform site visitors interested in life science news and information.