The interaction of riboflavin with a protein isolated from hen's egg white: A spectrofluorimetric study

Murthy, Usha S.; Podder, Santosh; Adiga, P. Radhakantha

doi:10.1016/0005-2795(76)90036-2

Cited by 45 publications

(21 citation statements)

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“…We found that more than 99% of RF molecules bind to the protein, as evidenced by the contribution to our transients of the free RF, which has a nanosecond lifetime (see below). The association constant was reported to be 10 7 -10 8 M Ϫ1 (18,19). The binding constant of RF with tryptophan alone in the buffer solution is much smaller, because we obtained no direct ET reaction up to 32 mM tryptophan; the quenching of excited-state flavins was observed at 230 mM indole in methanol solution (20).…”

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confidence: 86%

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Zhong

Zewail

2001

Proc. Natl. Acad. Sci. U.S.A.

219

222

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Flavoproteins can function as hydrophobic sites for vitamin B2 (riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: Ϸ100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)͞tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a chargeseparated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase. D ynamical processes in proteins play a crucial role in biological structure-function correlations (1, 2), and one such process is electron transfer (ET). In biological systems, ET reactions are ubiquitous (3, 4), especially in enzymes with redox reactions (5). Flavoproteins with flavin chromophores are examples of such enzymes and are involved in various catalytic processes (6-8). The understanding of ET reactions of flavins in proteins and their redox reactions is critical to the enzyme function.In this contribution, we report our femtosecond studies of ET dynamics of riboflavin (RF; vitamin B 2 ) in RF-binding protein (RfBP) and flavin-adenine-dinucleotide (FAD) in glucose oxidase (GOX) ( Fig. 1 and Scheme 1). High-resolution x-ray crystallographic structures of both proteins have recently been reported, and the binding structures of flavins in the proteins are well determined (9-11). RfBP is a globular monomeric protein of approximate dimensions 50 ϫ 40 ϫ 35 Å with a single polypeptide chain of 219 amino acids, nine disulfide bridges, six ␣-helices, and four series of discontinuous areas of ␤ structure.The ligand-binding domain of RfBP is a hydrophobic cleft, Ϸ20 Å wide and 15 Å deep. The binding of RF occurs in the cleft with...

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confidence: 86%

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Zhong

Zewail

2001

Proc. Natl. Acad. Sci. U.S.A.

219

222

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“…has not been identified. The crystal structure for the egg-white protein has been recently described [14] and provides a basis for earlier biochemical observations that indicated the essentiality of the disulfide bridges for high-affinity riboflavin binding [15], as well as the role of tryptophan residues present in the riboflavin-binding site of RCP [16]. The stretch of phosphoserine residues is present between two helices that are linked to the main riboflavin-binding core of the protein, therefore allowing the phosphopeptide to function autonomously in mediating receptor interaction [14].…”

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confidence: 99%

“…The presence of 18 cysteine residues in RCP, all involved in disulfide bridge formation [12] allows both the monitoring of the oxidation of the cysteine residues and the generation of riboflavin-binding activity simultaneously during the refolding. A sensitive and non-destructive method of monitoring riboflavin binding is to measure the quenching of riboflavin fluorescence on binding to the Apo protein [16]. The state of the free thiol content in RCP during refolding can also be measured [20] at the concentration of RCP used during refolding.…”

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confidence: 99%

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Pattanaik

Adiga

Visweswariah

1998

European Journal of Biochemistry

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Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.Keywords : riboflavin carrier protein; refolding; monoclonal antibodies.Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg white, yolk and serum of laying hens and other birds [1,2]. The egg-white protein is synthesized in the oviduct of the laying hen [3]. The liver, under the influence of estrogen, synthesizes serum RCP [3,4] which is then deposited through receptor-mediated uptake into the yolk of the developing egg [4]. RCP delivers riboflavin to the developing embryo in the egg, and birds that harbor a mutant RCP gene fail to lay eggs that hatch, as a result of insufficient deposition of the vitamin in the egg [5,6]. Egg-white RCP differs from the yolk and serum RCP only in the pattern of glycosylation [7] and removal of the C-terminal 11Ϫ13 amino acids in yolk RCP, presumably as a result of proteolytic cleavage during oocyte uptake [8]. A stretch of phosphoserine residues is present towards the C-terminal end of RCP [9]. We and others have shown the critical importance of these residues in mediating oocyte-receptor-binding and uptake of RCP during egg development [10,11].RCP is a complex protein with 18 cysteine residues all involved in disulfide-bridge formation [12]. Early studies had indicated that a single disulfide bridge appears to be critical for riboflavin binding [13], but the specific disulfide bridge involved ha...

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“…Since RF binds to RBP very strongly (K a = 10 8 M −1 according to Ref. 35), its washing time is much longer. Using Eqn.…”

Section: Resultsmentioning

confidence: 99%

Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection

Морозов¹,

Morozova²,

Johnson³

et al. 2003

Rapid Comm Mass Spectrometry

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Analysis of interactive networks between proteins and other molecular constituents is of paramount importance to delineate complex cellular processes. In order to facilitate this process, new technologies that allow rapid, high-throughput parallel screening, as well as identification of constituents, are necessary. A particularly powerful combination in this regard could be the use of multiprotein microarrays coupled with mass spectrometry (MS). In the initial step of the method development we applied MS to single-protein microarrays. We demonstrated that even a simplified version of the method allows rapid parallel label-free assay of specific protein interactions with multiple metabolites derived from complex artificial and natural mixtures. The microarrays fabricated by the electrospray deposition technique and cross-linked in glutaraldehyde vapor were brought into contact with droplets of solution containing either a natural extract of baker's yeast cells or an artificial cocktail of metabolites. After washing, the microarrays were placed into 75% methanol to denature proteins and release specifically bound metabolites. The eluates were then analyzed by electrospray ionization mass spectrometry (ESI-MS) to simultaneously detect all the metabolites bound. Such a procedure applied to ten different proteins demonstrated that 50-400 ng of cross-linked protein is enough to obtain ion intensities from metabolites that are well distinguishable above noise. The compatibility of microplates and different microarray designs with MS detection is discussed.

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The interaction of riboflavin with a protein isolated from hen's egg white: A spectrofluorimetric study

Cited by 45 publications

References 17 publications

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection

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The interaction of riboflavin with a protein isolated from hen's egg white: A spectrofluorimetric study

Cited by 45 publications

References 17 publications

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B 2 )-binding protein and in glucose oxidase enzyme

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B 2 )-binding protein and in glucose oxidase enzyme

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Parallel determination of multiple protein metabolite interactions using cell extract, protein microarrays and mass spectrometric detection

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Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme