Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies

Eftink, Maurice R.; Ghiron, Camillo A.

doi:10.1021/bi00648a035

Cited by 1,062 publications

(885 citation statements)

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“…Acrylamide quenching and quenching constant determination were performed as described previously (23)(24)(25) to investigate the degree of aqueous exposure of the sole tryptophan ( 50 Trp) present in both proSCP-2 and SCP-2. Briefly, fluorescence emission spectra of proSCP-2, SCP-2, or tryptophan (Trp) in the absence (F 0 ) or presence (F) of the small molecule quencher, acrylamide, were obtained by the following procedure.…”

Section: Acrylamide Quenching Of Tryptophan Fluorescencementioning

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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Section: Acrylamide Quenching Of Tryptophan Fluorescencementioning

confidence: 99%

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

et al. 2008

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“…The wavelength at the emission maximum for SPI was not varied by the addition of acrylamide, but was slightly shifted to a shorter wavelength by 3 nm for SP2 at the highest concentration. The ratio of the fluorescence intensity in the absence and presence of quencher was plotted against the concentration of quencher, according to the method reported by Eftink and Ghiron,20) as given in Fig. 5.…”

Section: Fluorescence Propertiesmentioning

confidence: 99%

Physicochemical Characteristics and Conformational Features of Alginate Lyase Isozymes fromTurbo cornutus

Muramatsu

Hashimoto

Takahashi

1984

Agricultural and Biological Chemistry

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“…Measurement of perturbation of intrinsic tryptophan fluorescence has proved to be a useful technique to monitor the binding of Ni2+ and other divalent metal ions to NikA. When excited at 290 nm, native NikA shows a fluorescence emission maximum at 342 nm, consistent with a moderate degree of burial of the nine tryptophans in the molecule (Eftink and Ghiron, 1976). On addition of Ni2', there was a small decrease in fluorescence and a shift in the emission maximum to 344 nm (Fig.…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of the Periplasmic Nickel‐Binding Protein NikA of Escherichia coli K12

Pina

Navarro

Mcwalter

et al. 1995

European Journal of Biochemistry

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The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pREl. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence.NikA binds one atom of nickellmolecule of protein with a dissociation constant (Kd) of less than 0.1 pM.Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 pM NiZ+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23000 molecules of NiWcell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.Keywords. nikA gene ; nickel binding ; periplasmic protein ; transport; overproduction.Bacterial cold-osmotic-shock-sensitive transport systems are complex permeases composed of a periplasmic substrate-binding receptor and a membrane-bound complex containing 2 -4 proteins (Ames, 1986). These permeases are energized by the hydrolysis of ATP, mediated by one or two of the proteins in the complex. The periplasmic substrate-binding proteins are the primary receptors for the transport systems specific for different small molecules such as sugars, amino acids, peptides and inorganic ions. In some cases, they also serve as the primary receptors for bacterial chemotaxis (Bourret et al., 1991;Furlong, 1987;Macnab, 1987). For example, in addition to its role in transport, the periplasmic maltose-binding protein, MBP, encoded by ma1E functions as the primary maltose chemoreceptor (Koiwai and Hayashi, 1979;Richarme, 1982). Generation of the chemotactic response to maltose requires a second protein, the chemotactic signal transducer Tar, which is also required for the chemotactic response to aspartate and to two repellents Ni2+ and Co2+ (Springer et al., 1977;Tso and Adler, 1974 Nickel has long been known as a heavy metal toxic to both eukaryotic and prokaryotic organisms (Barbich and Stotzky, 1983 ;Brown and Sunderman, 1988). Epidemiological studies have identified nickel as potentially carcinogenic and allergenic to humans (Dool et al., 1977; MennC et al., 1989). We demonstrated recently that nickel has an antagonistic effect on the fermentative growth of Escherichia coli . In E.coli the only known nickel-containing enzymes are the three hydrogenase isoenzymes whic...

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Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies

Cited by 1,062 publications

References 34 publications

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

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

Physicochemical Characteristics and Conformational Features of Alginate Lyase Isozymes fromTurbo cornutus

Purification and Characterization of the Periplasmic Nickel‐Binding Protein NikA of Escherichia coli K12

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