Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

Knubovets, Tatyana; Osterhout, John J.; Klibanov, Alexander M.

doi:10.1002/(sici)1097-0290(19990420)63:2<242::aid-bit13>3.0.co;2-n

Cited by 93 publications

(58 citation statements)

References 35 publications

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“…The characteristic tertiary structure of lysozyme in aqueous DMSO mixtures can be monitored with near-UV circular dichroism (CD) experiments ( Figure 6). 27,28 The spectra at DMSO volume fractions up to DMSO ) 0.6 resemble each other, while they are clearly different from the spectra for DMSO g 0.7. If we monitor the CD signal at a fixed wavelength as a function of DMSO , we see that the signal is enhanced up to DMSO ∼ 0.6, while it strongly decreases and eventually disappears at higher DMSO volume fractions.…”

Section: Resultsmentioning

confidence: 86%

“…Indeed, a reduction in the chemical shift dispersion in nuclear magnetic resonance (NMR) spectra of lysozyme in DMSO ) 0.5, 0.8, 0.9, and 1.0 indicates that several regions in the protein are more flexible as compared to that of the protein in DMSO ) 0. 1,27,28 We might speculate that DMSO does not unfold lysozyme, even though many H-bonds may be broken and some parts of the macromolecule are more flexible, until the stability of the region around the buried tryptophan residues is compromised. The UV-vis experiments of Hamaguchi et al support this idea.…”

Section: Resultsmentioning

confidence: 99%

“…While the effect of DMSO on the secondary and tertiary structure of lysozyme has been well documented, 1,[27][28][29][30] little is known about DMSO-induced changes in lysozyme size, shape, and interactions. In the following, we will provide the reader with a detailed description and in-depth discussion of the experimental results obtained in this study as well as previously reported results of several other groups taken from the literature.…”

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confidence: 99%

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DMSO-Induced Denaturation of Hen Egg White Lysozyme

et al. 2010

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We report on the size, shape, structure, and interactions of lysozyme in the ternary system lysozyme/DMSO/ water at low protein concentrations. Three structural regimes have been identified, which we term the "folded" (0 < DMSO < 0.7), "unfolded" (0.7 e DMSO < 0.9), and "partially collapsed" (0.9 e DMSO < 1.0) regime. Lysozyme resides in a folded conformation with an average radius of gyration of 1.3 ( 0.1 nm for DMSO < 0.7 and unfolds (average R g of 2.4 ( 0.1 nm) above DMSO > 0.7. This drastic change in the protein's size coincides with a loss of the characteristic tertiary structure. It is preceded by a compaction of the local environment of the tryptophan residues and accompanied by a large increase in the protein's overall flexibility. In terms of secondary structure, there is a gradual loss of R-helix and concomitant increase of -sheet structural elements toward DMSO ) 0.7, while an increase in DMSO at even higher DMSO volume fractions reduces the presence of both R-helix and -sheet secondary structural elements. Protein-protein interactions remain overall repulsive for all values of DMSO . An attempt is made to relate these structural changes to the three most important physical mechanisms that underlie them: the DMSO/water microstructure is strongly dependent on the DMSO volume fraction, DMSO acts as a strong H-bond acceptor, and DMSO is a bad solvent for the protein backbone and a number of relatively polar side groups, but a good solvent for relatively apolar side groups, such as tryptophan.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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DMSO-Induced Denaturation of Hen Egg White Lysozyme

et al. 2010

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“…Problems may arise, however, when the protein (partially) dissolves in the organic phase. The latter often results in unfolding [22,23]. In this study, lysozyme was loaded into porous microspheres by a simple solution immersing method without any harsh preparation conditions, such as the aqueous/organic interface produced by a water-in-oil microemulsion and the acute shearing strength brought by a high-speed homogenizer or a sonicater.…”

Section: Resultsmentioning

confidence: 99%

Preparation of “Open/closed” pores of PLGA-microsphere for controlled release of protein drug

et al. 2018

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Poly(D,L-lactic-co-glycolic acid) has been extensively used as a controlled release carrier for drug delivery due to its good biocompatibility, biodegradability, and mechanical strength. In this study, porous PLGA microspheres were fabricated by an emulsion-solvent evaporation technique using poly ethylene glycol (PEG) as an extractable porogen and loaded with protein (lysozyme) by suspending them in protein solution. For controlled release of protein, porous microspheres containing lysozyme were treated with water-miscible solvents in aqueous phase for production of pore-closed microspheres. The surface morphology of microspheres were investigated using scanning electron microscopy (SEM) for confirmation of its porous microstructure structure. Protein property after release was observed by enzymatic activity assay. The pore-closing process resulted in nonporous microspheres which exhibited sustained release patterns over an extended period.

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“…In the peptide-induced fluorescence quenching experiment, black 96-well microplates were filled with a final concentration of 100 µM of EIII in 1X PBS pH 7.4 containing the peptides of a varying concentrations (2,8,20 and 40 µM); and the samples were incubated at 25°C for 1 h. The final concentration of DMSO and DMF was fixed to 1%. To diminish the effects of DMSO and DMF on the tryptophan fluorescence of EIII in the peptide-induced fluorescence experiment, a reference control was prepared where 1% of each solvent was added to EIII without the presence of peptide.…”

Section: Methodsmentioning

confidence: 99%

Dengue Envelope Domain III-Peptide Binding Analysis <i>via</i> Tryptophan Fluorescence Quenching Assay

Baharuddin

Hassan

Othman

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λ max for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.Key words dengue virus type 2; envelope protein; tryptophan assay; fluorescence spectroscopy More than 2.5 million people are at risk of dengue infections in over 100 countries, with several hundred thousand cases of life threatening dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS) occurring annually.1) Currently, there is no commercially available vaccine or antiviral agent against dengue virus (DENV) infections. In the efforts towards developing an anti-viral treatment for dengue, a simple tryptophan fluorescence quenching screening assay aimed at identifying dengue EIII protein inhibitors was developed.DENV is a positive-sense, single stranded RNA virus belonging to the Flaviviridae family. The DENV consists of four antigenically distinct serotypes (serotypes 1-4). The glycosylated envelope (E) protein is a type 1 integral membrane protein, and makes up the major structural protein present on the surface of the mature dengue virions. It plays an important role in viral infection via virus attachment to cell-surface receptors and mediates viral-cell membrane fusion. The E protein is the major target for protective antibodies against dengue.2) It has been demonstrated that the mature DENV E protein in the pre-fusion state forms homodimers in an antiparallel manner (head to tail orientation), which transforms to its trimeric form in the post-fusion state.2) The stability and function of the DENV E protein during conformational transition from dimeric to trimeric form is controlled by the change in pH. Each monomer is folded in three distinct domains, with the central domain (domain I) bordered by the immunoglobulin-like C-terminal domain (domain III) and the dimer...

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Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

Cited by 93 publications

References 35 publications

DMSO-Induced Denaturation of Hen Egg White Lysozyme

DMSO-Induced Denaturation of Hen Egg White Lysozyme

Preparation of “Open/closed” pores of PLGA-microsphere for controlled release of protein drug

Dengue Envelope Domain III-Peptide Binding Analysis <i>via</i> Tryptophan Fluorescence Quenching Assay

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