(M1130-09-57) Revisiting UV Spectroscopy for Real-Time Monitoring of Reversible Protein Unfolding

Purpose: The kinetics` of protein denaturation is of interest due to protein instability when exposed to chemical and physical stress. Limitations of current measurement techniques include a lag between sample prep and analysis, and volume, mixing and concentration constraints. UV spectroscopy can detect changes in protein folding due to spectral differences between native and denatured protein forms^1-3. In this work we show how 2^nd derivative UV can monitor protein unfolding/folding in real-time. Reversible unfolding of BSA, a model protein, induced by sodium dodecyl sulphate (SDS) and hydroxypropyl-β-cyclodextrin (HP-β-CD) ^4-6 were studied.

Methods: Experiments were carried out using Pion Rainbow R6 fiber optic spectrometer featuring Zero Intercept Method (ZIM) analysis built into AuPro Software and a Chirascan™ Plus circular dichroism (CD) spectrometer Spectral changes of BSA (3 µM) were monitored at pH 7.4 in 50 mM phosphate buffer spiking solution with stocks of SDS and HP-β-CD to progressively induce protein unfolding and refolding. The 2^nd derivative absorption profile of UV active compounds crosses the wavelength coordinate at specific wavelengths (λ_ZIM), referred to as ZIM points. While the spectral magnitude reflects changes in concentration, the λ_ZIM stays constant. However, when a sample undergoes structural changes, the λ_ZIM shifts correlating with the fraction of the new absorbing species. Caffeine and tryptophan were used as control samples. CD spectra of BSA were collected within 300-190 nm at 60 nm/min, 1.0 nm bandwidth. Mean residue ellipticity (MRE) at 222 nm was determined and secondary structure (alpha helix, beta sheet and random coil content) calculated using CDNN software.

Results: λ_ZIM of BSA, caffeine and tryptophan were monitored in the presence of SDS ranging 0 – 35 mM. The ZIM point of BSA (λ_ZIM ^native) measured in the absence of SDS at 289.6 nm was sequentially shifting 0.1 – 0.3 nm, reaching a minimum λ_ZIM ^unfolded 288.5 nm at 5 mM SDS. In the presence of HP-β-CD (concentration range 2 – 16 mM), λ_ZIM^unfolded shifted in the opposite direction, showing that the reversibility of the unfolding process was captured by the λ_ZIM method. ZIMs of caffeine (λ_ZIM 286.4 nm) and tryptophan (λ_ZIM 292.3 nm) stayed constant in the same conditions, fluctuating within 0.03 – 0.06 nm. Changes of the BSA λ_ZIM were compared to the mean residue ellipticity parameter obtained by Far-UV CD and showed similarities between methods as shown in figures 1 and 2.

Conclusion: Our results demonstrate the implementation of UV spectroscopy for monitoring protein structural changes at timescales from 2 seconds to days. This method can be applied to a broader variety of proteins, although potential limitations are yet to be studied. Fiber optic derivative UV spectroscopy can be employed as a labor-saving tool to monitor protein structural changes, allowing stable and flexible data collection, as well as fast and reliable data processing.

References: 1. Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation–p interactions 1 Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation–p interactions Laura Lucas, Baran Ersoy, Lisa Kueltzo, Sangeeta Joshi, Duane Brandau, Nagarajan Thyagarajapuram, Laura Peek, Russell Middaugh Protein Sci. 2006 Oct; 15(10): 2228–2243.
2. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore Jan M Antosiewicz, David Shugar Biophys Rev 2016 Jun;8(2):151-161
3. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 2: selected applications Jan M Antosiewicz , David Shugar, Biophys Rev. 2016 Jun;8(2):163-177.
4. DSC studies on bovine serum albumin denaturation Effects of ionic strength and SDS concentration C. Giancola, C De Sena, D. Fessas, G Graziano, G Barone, Int J Biol Macromol 1997 Jun;20(3):193-204
5. Protein unfolding and subsequent refolding: a spectroscopic investigation Uttam Anand, Chandrima Jash and Saptarshi Mukherjee, Phys. Chem. Chem. Phys., 2011, 13, 20418–20426
6. Reversibility in protein folding: effect of β-cyclodextrin on bovine serum albumin unfolded by sodium dodecyl sulphate. Uttam Anand, Saptarshi Mukherjee, PCCP, 2013, 15(23)

BSA-unfolding: CDNN determined beta sheet content (left axes) and the drift of ZIM 289 (right axis) as a function of SDS concentration

BSA-refolding: CDNN determined beta sheet content (left axes) and the drift of ZIM 289 (right axis) as a function of HP-β-CD concentration