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Investigating native state fluorescence emission of Immunoglobulin G using polarized Excitation Emission Matrix (pEEM) spectroscopy and PARAFAC
摘要： Intrinsic ?uorescence spectroscopy (IFS) measurements for protein structural analysis can be enhanced by the use of anisotropy resolved multidimensional emission spectroscopy (ARMES). ARMES attempts to overcome the tryptophan (Trp) and tyrosine (Tyr) spectral overlap problem and resolve emitting components by combining anisotropy measurements with chemometric analysis. Here we investigate for the ?rst time the application of polarized excitation-emission matrix (pEEM) measurements and Parallel Factor (PARAFAC) analysis to study IFS from an Immunoglobulin G (IgG) type protein, rabbit IgG (rIgG), in its native state. Protein IFS is a non-trilinear system primarily because of F€orster resonance energy transfer (FRET). Non-trilinearity is also caused by inner ?lter effects, and Rayleigh/Raman scattering, both of which can be corrected by data pre-processing. However, IFS FRET cannot be corrected for, and thus here we carefully evaluated the impact of various different data pre-processing methods on IFS data which used for PARAFAC. Care must be taken with data pre-processing and interpolation, as both had an impact on PARAFAC modelling and the recovered anisotropy values, with residual shot noise from the Rayleigh scatter which overlapped the emission blue edge being the root cause. pEEM spectra from thawed rIgG solutions (15–35 (cid:1)C temperature range) were collected with an expectation being that this temperature range should cause suf?cient emission variation to facilitate component resolution but without major structural changes. However, the only signi?cant changes observed were of the overall intensity due to thermal motion induced quenching and this was con?rmed by the PARAFAC scores. PARAFAC resolved one major component (>99%) for the emission data (polarized and unpolarized) which mostly represented the large Tyr-to-Trp hetero-FRET process, with a second, very weak component (<1%) apparently a contribution from directly excited Trp emission. PARAFAC scores recovered from normalized pEEM data showed minimal change which was further proof for negligible structural change. The results of this study serves as the starting point for the use of PARAFAC analysis of IFS from IgG type proteins and important processes such as denaturation and aggregation.
关键词： Multidimensional,Fluorescence,PARAFAC,Anisotropy,Protein,Spectroscopy,Immunoglobulin G
更新于2025-11-14 15:32:45
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Photo-controlled reversible binding between the protein A-derived Z domain and immunoglobulin G
摘要： Photoisomerization of the trans and cis isomers of azobenzene derivatives has been used to control the function of biomolecules in a reversible and non-destructive manner. In this study affibody molecules, representing a class of small, helical proteins that can be engineered for binding to a wide range of target proteins, have been investigated by the incorporation of a photoswitchable azobenzene derivative in the molecule. Three different Z domain variants were produced by solid phase peptide synthesis and conjugated by thiol-directed chemistry to an azobenzene-based photoswitch. The proteins were screened for binding to and light elution from an IgG-sepharose affinity column. One of tested Z variants, ZC3, showed efficient binding to the column and could be eluted by irradiation with light at 400 nm. In a reverse affinity chromatography assay, where the ZC3 variant was coupled to sepharose, human IgG1 could be captured to the column and partially eluted by light. Further studies of the azobenzene-conjugated ZC3 domain by surface plasmon resonance (SPR) confirmed the high affinity binding to IgG, and circular dichroism (CD) spectroscopy showed that the protein has a high alpha-helical secondary structure content.
关键词： immunoglobulin G,affibody molecule,photoswitch,Z domain,affinity chromatography,azobenzene
更新于2025-09-19 17:13:59
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Orientation-Controlled Bioconjugation of Antibodies to Silver Nanoparticles
摘要： Here we report on the use of heterobifunctional cross-linkers (HBCLs) to control the number, orientation, and activity of immunoglobulin G antibodies (Abs) conjugated to silver nanoparticles (AgNPs). A hydrazone conjugation method resulted in exclusive modification of the polysaccharide chains present on the fragment crystallizable region of the Abs, leaving the antigen-binding regions accessible. Two HBCLs, each having a hydrazide terminal group, were synthesized and tested for effectiveness. The two HBCLs differed in two respects, however: (1) either a thiol or a dithiolane group was used for attachment to the AgNP; and (2) the spacer arm was either a PEG chain or an alkyl chain. Both cross-linkers immobilized 5 ± 1 Abs on the surface of each 20-nm-diameter AgNP. Electrochemical results, obtained using a half-metalloimmunoassay, proved that Abs conjugated to AgNPs via either of the two HBCLs were 4 times more active than those conjugated by the more common physisorption technique. This finding confirmed that the HBCLs exerted orientational control over the Abs. We also demonstrated that the AgNP-HBCL-Ab conjugates were stable and active for at least 2 weeks. Finally, we found that the stability of the HBCLs themselves was related to the nature of their spacer arms. Specifically, the results showed that the HBCL having the alkyl chain is chemically stable for at least 90 days, making it the preferred cross-linker for bioassays.
关键词： alkyl chain,antigen-binding regions,heterobifunctional cross-linkers,PEG chain,hydrazone conjugation,physisorption technique,immunoglobulin G antibodies,silver nanoparticles,half-metalloimmunoassay,fragment crystallizable region
更新于2025-09-12 10:27:22