Circular dichroism for secondary structure determination of proteins with unfolded domains using a self-organising map algorithm SOMSpec

Olamoyesan, Adewale; Ang, Dale L.; Rodger, Alison

doi:10.1039/d1ra02898g

Cited by 17 publications

(7 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In aggregate, the data demonstrate the ability of the hydrogels to release DOX in its active form through photothermal modulation. These data together with prior reports of the photothermally induced release of active proteins from hydrogels − and of protein stability up to 60 °C , bode well for the potential of our hydrogels as a delivery system for a wide array of therapeutic agents.…”

Section: Resultssupporting

confidence: 67%

Photothermal Modulation of Dynamic Covalent Poly(ethylene glycol)/PEDOT Composite Hydrogels for On-Demand Drug Delivery

Thapa,

FitzSimons,

Otakpor

et al. 2023

ACS Appl. Mater. Interfaces

View full text Add to dashboard Cite

Hydrogels are cross-linked three-dimensional polymer networks that have tissue-like properties. Dynamic covalent bonds (DCB) can be utilized as hydrogel cross-links to impart injectability, self-healing ability, and stimuli responsiveness to these materials. In our research, we utilized dynamic thiol-Michael bonds as cross-links in poly(ethylene glycol) (PEG)-based hydrogels. Because the equilibrium of the reversible, exothermic thiol-Michael reaction can be modulated by temperature, we investigated the possibility of using thermal and photothermal stimuli to modulate the gel-to-sol transition of these materials with the aim of developing an on-demand pulsatile cargo release system. For this purpose, we incorporated poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles within the hydrogel to facilitate photothermal modulation using near-infrared light. PEDOT nanoparticles of 50 nm in diameter and with strong near-infrared absorption were prepared by oxidative emulsion polymerization. We then used Michael addition of thiol−ene pairs from 4-arm PEG-thiol (PEG-SH) and 4-arm PEG-benzylcyanoacetamide (PEG-BCA) to form dynamically cross-linked hydrogels. PEDOT nanoparticles were entrapped in situ to form Gel/PEDOT composites. Rheology and inverted tube test studies showed that the gel-to-sol transition occurred at 45−50 °C for 5 wt % gels and that this transition could be tailored by varying the wt % of the polymer precursors. The hydrogels were found to be capable of self-healing and being injected with a clinically relevant injection force. Bovine serum albumin-fluorescein isothiocyanate (BSA-FITC), a fluorescently labeled protein, was then loaded into the Gel/PEDOT as a therapeutic mimic. Increased release of BSA-FITC upon direct thermal stimulation and photothermal stimulation with an 808 nm laser was observed. Pulsatile release of BSA-FITC over seven cycles was demonstrated. MTS and live−dead assays demonstrated that Gel/PEDOT was cytocompatible in MDA-MB-231 breast cancer and 3T3 fibroblast cell lines. Further studies demonstrated that the encapsulation and laser-triggered release of the chemotherapeutic agent doxorubicin (DOX) could also be achieved. Altogether, this work advances our understanding of the temperature-dependent behavior of a dynamic covalent hydrogel, Gel/PEDOT, and leverages that understanding for application as a photothermally responsive biomaterial for controlled release.

show abstract

Section: Resultssupporting

confidence: 67%

Photothermal Modulation of Dynamic Covalent Poly(ethylene glycol)/PEDOT Composite Hydrogels for On-Demand Drug Delivery

Thapa,

FitzSimons,

Otakpor

et al. 2023

ACS Appl. Mater. Interfaces

View full text Add to dashboard Cite

show abstract

“…In particular, shifts of wavelength maxima and loss of spectral intensity at high wavenumbers or low wavenumbers indicate, respectively, low helix and low sheet content in the prediction. We also found that proteins such as lysozyme and papain which have β II characteristics in their CD spectra (Whitmore et al, 2011;Olamoyesan et al, 2021) have helix-like IR spectra. Finally, proteins with prosthetic groups which absorb in the Amide I region such as flavins and hemes may also cause errors in secondary structure predictions.…”

Section: Discussionmentioning

confidence: 58%

“…The problem classes are again 1) high helix content proteins especially those with papain (S19) and/or lysozyme (S12) among their BMUs (the helix content is significantly underestimated), 2) β II proteins in particular trypsin (S24) and chymotrypsin (S25), and 3) the largely unfolded bungarotoxin (S30). Interestingly, both lysozyme and papain CD spectra (Whitmore et al, 2011;Olamoyesan et al, 2021) have β II characteristics suggesting this is a key to the problems with the first two types.…”

Section: Solid-state Protein Ir Spectra Loovmentioning

confidence: 99%

SOMSpec as a General Purpose Validated Self-Organising Map Tool for Rapid Protein Secondary Structure Prediction From Infrared Absorbance Data

et al. 2022

Self Cite

View full text Add to dashboard Cite

A protein’s structure is the key to its function. As protein structure can vary with environment, it is important to be able to determine it over a wide range of concentrations, temperatures, formulation vehicles, and states. Robust reproducible validated methods are required for applications including batch-batch comparisons of biopharmaceutical products. Circular dichroism is widely used for this purpose, but an alternative is required for concentrations above 10 mg/mL or for solutions with chiral buffer components that absorb far UV light. Infrared (IR) protein absorbance spectra of the Amide I region (1,600–1700 cm−1) contain information about secondary structure and require higher concentrations than circular dichroism often with complementary spectral windows. In this paper, we consider a number of approaches to extract structural information from a protein infrared spectrum and determine their reliability for regulatory and research purpose. In particular, we compare direct and second derivative band-fitting with a self-organising map (SOM) approach applied to a number of different reference sets. The self-organising map (SOM) approach proved significantly more accurate than the band-fitting approaches for solution spectra. As there is no validated benchmark method available for infrared structure fitting, SOMSpec was implemented in a leave-one-out validation (LOOV) approach for solid-state transmission and thin-film attenuated total reflectance (ATR) reference sets. We then tested SOMSpec and the thin-film ATR reference set against 68 solution spectra and found the average prediction error for helix (α + 310) and β-sheet was less than 6% for proteins with less than 40% helix. This is quantitatively better than other available approaches. The visual output format of SOMSpec aids identification of poor predictions. We also demonstrated how to convert aqueous ATR spectra to and from transmission spectra for structure fitting. Fourier self-deconvolution did not improve the average structure predictions.

show abstract

“…This reduction in peak intensity at 265 nm is proportional to the distortion due to a decrease in DNA/RNA base stacking. 28 Comparison of the CD spectrum of hnRNPB1 (1−195) and hnRNPB1 (1−278) shows the presence of a disordered structure in the LCD, which is evident by the shift of a negative maximum at 217 nm (β-sheet) toward 200 and 195 nm positive peak, which is also smaller than that expected 29 (Figure S15B). This region of LCD (195−278) used in our studies lacks structural order, evidenced by previous structural studies.…”

Section: Biochemistrymentioning

confidence: 99%

Interaction of hnRNPB1 with Helix-12 of hHOTAIR Reveals the Distinctive Mode of RNA Recognition That Enables the Structural Rearrangement by LCD

Kumar,

Daripa,

Maiti

et al. 2023

Biochemistry

View full text Add to dashboard Cite

The lncRNA human Hox transcript antisense intergenic RNA (hHOTAIR) regulates gene expression by recruiting chromatin modifiers. The prevailing model suggests that hHOTAIR recruits hnRNPB1 to facilitate intermolecular RNA−RNA interactions between the lncRNA HOTAIR and its target gene transcripts. This B1-mediated RNA−RNA interaction modulates the structure of hHOTAIR, attenuates its inhibitory effect on polycomb repression complex 2, and enhances its methyl transferase activity. However, the molecular details by which the nuclear hnRNPB1 protein assembles on the lncRNA HOTAIR have not yet been described. Here, we investigate the molecular interactions between hnRNPB1 and Helix-12 (hHOTAIR). We show that the low-complexity domain segment (LCD) of hnRNPB1 interacts with a strong affinity for Helix-12. Our studies revealed that unbound Helix-12 folds into a specific base-pairing pattern and contains an internal loop that, as determined by thermal melting and NMR studies, exhibits hydrogen bonding between strands and forms the recognition site for the LCD segment. In addition, mutation studies show that the secondary structure of Helix-12 makes an important contribution by acting as a landing pad for hnRNPB1. The secondary structure of Helix-12 is involved in specific interactions with different domains of hnRNPB1. Finally, we show that the LCD unwinds Helix-12 locally, indicating its importance in the hHOTAIR restructuring mechanism.

show abstract

Circular dichroism for secondary structure determination of proteins with unfolded domains using a self-organising map algorithm SOMSpec

Abstract: Circular dichroism secondary structure fitting by analysing derandomized spectra using the SOMSpec approach then regenerating data for the original spectrum.

Cited by 17 publications

References 28 publications

Photothermal Modulation of Dynamic Covalent Poly(ethylene glycol)/PEDOT Composite Hydrogels for On-Demand Drug Delivery

Photothermal Modulation of Dynamic Covalent Poly(ethylene glycol)/PEDOT Composite Hydrogels for On-Demand Drug Delivery

SOMSpec as a General Purpose Validated Self-Organising Map Tool for Rapid Protein Secondary Structure Prediction From Infrared Absorbance Data

Interaction of hnRNPB1 with Helix-12 of hHOTAIR Reveals the Distinctive Mode of RNA Recognition That Enables the Structural Rearrangement by LCD

Contact Info

Product

Resources

About