The use of fluorescence methods to monitor unfolding transitions in proteins

Eftink, Maurice R.

doi:10.1016/s0006-3495(94)80799-4

Cited by 503 publications

(443 citation statements)

References 68 publications

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“…c −Control is formulated 3D8 scFv without stabilizer. that the tryptophan residue of 3D8 scFv is close to the surface of the protein and is partly exposed to external environments (Sharma and Kalonia, 2003;Eftink, 1994). In cases of heparin, mannitol and PEG, the emission wavelength maxima were around 337 nm.…”

Section: Additivementioning

confidence: 94%

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Son

Lee

Joung

et al. 2009

International Journal of Pharmaceutics

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Section: Additivementioning

confidence: 94%

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Son

Lee

Joung

et al. 2009

International Journal of Pharmaceutics

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“…Quenching of tryptophan fluorescence by the small molecule acrylamide is an effective method of further analyzing the surface and aqueous exposure of Trp in Trp-containing proteins (37,42). To this end, 50 Figure 4A).…”

Section: Aqueous Exposure Of the 50 Trp Residue In Proscp-2 And Scp-2mentioning

confidence: 99%

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

et al. 2008

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Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2's affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.Keywords sterol carrier protein-2; presequence; cholesterol; peroxin; peroxisome Sterol carrier protein-2 (SCP-2) 1 is a ubiquitous, soluble protein present at highest level in tissues involved in cholesterol uptake (intestine), oxidation (liver, steroidogenic cells), and/or elimination (liver) (reviewed in ref 1). SCP-2 exhibits high (nanomolar K d values) affinity for

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“…During denaturation, the emission maximum for proteins containing tryptophan is usually shifted from shorter wave- lengths to longer wavelengths [22,23]. Although, the unfolding of CK during thermal denaturation is signi®cantly a ected by the presence of Mg 2+ (Fig.…”

Section: Discussionmentioning

confidence: 99%

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

Cao

Luo

Zhou

1999

The International Journal of Biochemistry & Cell Biology

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The e ect of Mg 2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg 2+ concentrations. Increasing the Mg 2+ concentration in the denatured system signi®cantly enhanced the inactivation and unfolding of creatine kinase during thermal denaturation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 478C the increased free Mg 2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the e ect of Mg 2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with di erent concentrations of Mg 2+ did not change the enzymatic activity. The¯uorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 478C for 10 min, while the presence of 3 mM Mg 2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg 2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 478C. The potential biological signi®cance of these results are discussed. #

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The use of fluorescence methods to monitor unfolding transitions in proteins

Cited by 503 publications

References 68 publications

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

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