Tryptophan Fluorescence Of Protein Research Articles

Dark nanodiscs for evaluating membrane protein thermostability by differential scanning fluorimetry

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the “dark” nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.

Effects of resveratrol on lipid and protein co-oxidation in fish oil-enriched whey protein isolate emulsions

In this study, the impact of resveratrol (RES) on co-oxidation of lipid and protein in a fish oil-fortified whey protein isolate (WPI) emulsion was investigated. Oil-in-water (O/W) emulsions containing 1% fish oil, 6 mg/mL of WPI and RES (0.08 ~ 2 mM) were oxidatively stressed using a Fenton system at 25 °C for 24 h. The incorporation of RES significantly suppressed lipid oxidation (TBARS) and protein carbonylation. Oxidation-induced decrease on protein sulfhydryl content and surface hydrophobicity were partially attenuated by RES, but protein tryptophan fluorescence was further decreased with the increased concentration of RES. Visualization of protein patterns and MDA-bound protein suggested that RES is capable of inhibiting protein modification induced by secondary products of lipid oxidation. Significant decrease in protein digestibility under oxidizing condition was also mitigated by RES. Our study contributes to the exploration of complicated interactions between oxidized lipids and proteins when phenolic compounds are present.

Dye-protein interactions between Rhodamine B and whey proteins that affect the photoproperties of the dye

In fluorescence labeling, proteins are usually assayed to minimize the impact of fluorophores on their intrinsic structure and functions, however, the effect of the protein itself on the fluorescence properties of its attached fluorophore has not been studied at length. In this paper, the fluorescence properties of the ubiquitously used Rhodamine B (RhB) were investigated in the presence of whey proteins, particularly at alkaline conditions. Results reveal the existence of dye-protein interactions whether the dye is free or covalently linked to proteins. Alkali was able to destroy these interactions, while base-denaturation of proteins also occurred that complicates the dye-protein interactions. In addition, RhB was found able to quench the tryptophan (Trp) fluorescence of whey proteins, leading to the formation of nonfluorescent Trp-RhB complex. This stable Trp-RhB complex might explain the irreversible binding of RhB upon thermal-induced whey protein aggregates. This finding could suggest a cheaper alternative of labeling on proteins.

Interaction of Cp6-his and Cp6 with bovine serum albumin and liver microsomes: Spectroscopic and molecular docking studies

The conjugation of photosensitizer with a molecule specific to receptors over-expressed on cancer cells is a promising approach for improving tumor selectivity in photodynamic therapy (PDT). Since chemical nature of the photosensitizer is also affected by conjugation, this can have significant influence on its transport by serum proteins and clearance via liver microsomal enzyme system. In this report, the interaction of chlorin p6-histamine conjugate (Cp6-his) and free chlorin p6 (Cp6) with bovine serum albumin (BSA) and mouse liver microsomes was assessed by measuring the quenching of intrinsic tryptophan (Trp) fluorescence of proteins. The effect of photosensitizer binding on the activity of microsomal NADPH-cytochrome P-450 and NADH-cytochrome b5 reductase was also determined. The formation of protein carbonyl and lipid peroxides was measured to assess the photodynamic damage to proteins and lipids respectively. Further, the binding interaction between Cp6-his conjugate and parent Cp6 with BSA and cytochrome P-450 was validated using in-silico molecular docking approach. Results show that as compared to free Cp6, the affinity of conjugate to bind with serum albumin is 4 orders of magnitude less, which was also corroborated with finding of docking studies. In contrast, with microsomal proteins the binding affinity of Cp6-his was observed to be lower by 2 orders of magnitude as compared to that for Cp6. Further, the inhibition of the activities of microsomal enzymes induced by Cp6 conjugate and free Cp6 was nearly same (~15-20%). Measurements on quenching of photosensitizer fluorescence by iodide ions revealed that conjugate localized slightly deeper in microsomal membrane as compared to free Cp6 consistent with the slight difference in lipophilicity of the two photosensitizers. The amount of photodynamic damage to BSA induced by Cp6-his was significantly less as compared to that for Cp6, whereas no difference was observed in damage induced to microsomal protein and lipid. These results suggest that conjugation of Cp6 to histamine affects its binding to albumin but doses not significantly alters its interaction with microsomal enzyme system.

Genetic variation and potential for genetic improvement of cuticle deposition on chicken eggs

BackgroundThe cuticle is an invisible glycosylated protein layer that covers the outside of the eggshell and forms a barrier to the transmission of microorganisms. Cuticle-specific staining and in situ absorbance measurements have been used to quantify cuticle deposition in several pure breeds of chicken. For brown eggs, a pre-stain and a post-stain absorbance measurement is required to correct for intrinsic absorption by the natural pigment. For white eggs, a post-stain absorbance measurement alone is sufficient to estimate cuticle deposition. The objective of the research was to estimate genetic parameters and provide data to promote adoption of the technique to increase cuticle deposition and reduce vertical transmission of microorganisms.ResultsFor all pure breeds examined here, i.e. Rhode Island Red, two White Leghorns, White Rock and a broiler breed, the estimate of heritability for cuticle deposition from a meta-analysis was moderately high (0.38 ± 0.04). In the Rhode Island Red breed, the estimate of the genetic correlation between measurements recorded at early and late times during the egg-laying period was ~ 1. There was no negative genetic correlation between cuticle deposition and production traits. Estimates of the genetic correlation of cuticle deposition with shell color ranged from negative values or 0 in brown-egg layers to positive values in white- or tinted-egg layers. Using the intrinsic fluorescence of tryptophan in the cuticle proteins to quantify the amount of cuticle deposition failed because of complex quenching processes. Tryptophan fluorescence intensity at 330 nm was moderately heritable, but there was no evidence of a non-zero genetic correlation with cuticle deposition. This was complicated furthermore by a negative genetic correlation of fluorescence with color in brown eggs, due to the quenching of tryptophan fluorescence by energy transfer to protoporphyrin pigment. We also confirmed that removal of the cuticle increased reflection of ultraviolet wavelengths from the egg.ConclusionsThese results provide additional evidence for the need to incorporate cuticle deposition into breeding programs of egg- and meat-type birds in order to reduce vertical and horizontal transmission of potentially pathogenic organisms and to help improve biosecurity in poultry.

Myeloperoxidase-Induced Oxidation of Albumin and Ceruloplasmin: Role of Tyrosines.

Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.

Exploring the breakdown of dairy protein gels during in vitro gastric digestion using time-lapse synchrotron deep-UV fluorescence microscopy

A novel time-lapse synchrotron deep-UV microscopy methodology was developed that made use of the natural tryptophan fluorescence of proteins. It enabled the monitoring in situ of the microstructural changes of protein gels during simulated gastric digestion. Two dairy gels with an identical composition, but differing by the coagulation mode, were submitted to static in vitro gastric digestion. The kinetics of gel particle breakdown were quantified by image analysis and physico-chemical analyses of digesta. The results confirm the tendency of rennet gels, but not acid gels, to form compact protein aggregates under acidic conditions of the stomach. Consequently, the kinetics of proteolysis were much slower for the rennet gel, confirming the hypothesis of a reduced pepsin accessibility to its substrate. The particle shapes remained unchanged and the disintegration kinetics followed an exponential trend, suggesting that erosion was the predominant mechanism of the enzymatic breakdown of dairy gels in these experimental conditions.

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ1 = 6–7 ns, τ2 = 2.0–2.3 ns, and τ3 ≤ 0.1 ns (the native state), and τ1 = 4.4–4.6 ns, τ2 = 1.7–1.8 ns, and τ3 ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.

WW domain folding complexity revealed by infrared spectroscopy.

Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.

Static quenching of tryptophan fluorescence in proteins by a dioxomolybdenum(VI) thiolate complex

Abstract The binding of cis-dioxobis(dithiocarbamato) molybdenum(VI) with the proteins bovine serum albumin, human serum albumin, lysozyme, and free tryptophan was studied using fluorescence spectroscopy and Stern–Volmer kinetics. The quenching of tryptophan fluorescence was determined to be static with binding constants on the order of 104–105 M−1, and with a binding site number of one. The interaction was studied over a range of temperatures, and the binding was found to be exothermic with a negative change in entropy. Quantum chemical calculations were also conducted to identify optimal spatial contacts and the resulting energetic contributions between the complex and free tryptophan.

Thymoquinone inhibits microtubule polymerization by tubulin binding and causes mitotic arrest following apoptosis in A549 cells

Microtubule-Targeting agents (MTA) are indispensable for cancer therapeutics. We here report thymoquinone (TQ) as a new MTA that already has been appreciated for its anticancer effects. TQ induced G2/M cell cycle arrest in human non-small lung epithelial cells (A549) and majority of arrested cells were in mitosis. TQ depolymerized the microtubule (MT) network and disrupted mitotic spindle organization of A549 cells. MT depolymerization by TQ was followed by apoptosis and subsequent loss in cell viability (IC50 value of ∼10 μM). Interestingly, TQ didn't affect the MT network of normal HUVEC cells at and below the IC50 concentration for A549 cells. TQ also inhibited tubulin polymerization in cell-free system with an IC50 of 27 μM and bound to tubulin heterodimers at a single site with a dissociation constant of 1.19 μM at 25 °C. Binding of TQ to tubulin quenched the tryptophan fluorescence of protein in a time-dependent manner. The TQ-tubulin binding kinetics was biphasic in nature and equilibrated in 30 min. TQ competed with colchicine for tubulin binding with a Ki of 1.9 μM as determined by modified Dixon plot analysis, this suggests TQ may bind tubulin at or near the colchicine binding site and in silico modeling study supported that. Our results establish a novel antimitotic mechanism of TQ by its direct binding to tubulin-MT network in A549 cells.

Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10(2) M(-1) (free Trp) to 10(10) M(-1) (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.

Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

Static and Dynamic Quenching of Tryptophan Fluorescence in Various Proteins by a Chromium (III) Complex

ABSTRACT The interaction of a chromium (III) complex, (R,R)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexane-diaminochromium (III), with human serum albumin, bovine serum albumin, lysozyme, and free tryptophan was studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants were calculated using Stern-Volmer kinetics. The complex bound more tightly to the serum albumins than to lysozyme or free tryptophan, but only one binding site was determined in all systems. The interaction was also determined to be thermodynamically favorable, and the binding constants were on the order of 103–106. The fluorescence quenching was static in nature with Forster distances in the 1.8–2.0 nm range.

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein–ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.

Tryptophan Fluorescence from G. Weber to the Present

Gregorio Weber (Symposium on Light and Life, W.D. McElroy and Glass, B. Eds., 1961) described the fluorescence of tryptophan in proteins. Since that time more than 12,000 papers have appeared related to tryptophan fluorescence. It is used to measure the folding of proteins and the interaction of proteins with each other, with nucleic acids, membranes and small molecules. Time-resolved fluorescence studies have revealed details regarding the excited-state of tryptophan in proteins. Ground-state and excited-state heterogeneity influence the fluorescence and its relation to protein conformation. Excited-state electron transfer, proton transfer, energy transfer, solvent and protein relaxation are among the processes that have been implicated in tryptophan fluorescence in proteins The availability of new experimental, computational and theoretical methods suggest that there are now opportunities for using tryptophan fluorescence for probing protein structure and dynamics. Femtosecond time-resolved techniques and molecular dynamics computations have been of particular value and should provide new information about folded and unfolded structural regions in proteins.

Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies

Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3′,4′-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive ‘two color’ fluorescence signatures and fairly high fluorescence anisotropy ( r = 0.12–0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the fluorescence studies were ≈ 4.00 × 10 4 M − 1 and 9.83 × 10 3 M − 1 for fisetin and 3-HF respectively. Specific interactions with HbA were further confirmed from flavonoid-induced static quenching of the protein tryptophan fluorescence as indicated by: (a) bimolecular quenching constant K q ≫ diffusion controlled limit (b) closely matched values of Stern–Volmer quenching constant and binding constant (c) τ o/τ ≈ 1 (where τ o and τ are the unquenched and quenched tryptophan fluorescence lifetimes respectively). Molecular docking and electrostatic surface potential calculations reveal contrasting binding modes of fisetin and 3-HF with HbA.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Biophysical Characterization of Effect of Zinc on Eukaryotic Translation Initiation Factor 4B

Eukaryotic translation initiation factor 4B promotes RNA dependent ATP hydrolysis and ATP dependent RNA helicase activity of eIF4A and eIF4F. It has recently been reported that eIF4B also organizes assembly of the RNA, eIFiso4G, eIF4A, and poly‐(A) binding protein and zinc enhances the RNA binding and interaction with PABP. By the use of fluorescence spectroscopy, native protein tryptophan fluorescence, and fluorescence labeled RNA as internal molecular probes we are characterizing these interactions. We previously reported that zinc binds tightly with eIF4B, Kd of 19.7 nM (J Biol Chem. 2008 Dec 26; 283(52):36140–53). The Kd of binding of eIF4B with 20mer poly (A) RNA is 77 ± 7 nM and in presence of zinc it lowers down to 45 ± 3 nM. There was no significant difference between the binding of eIF4B full length and eIF4B (320–527) deletion mutant with 20mer poly(A) RNA. Circular dichroism showed that addition of zinc resulted in a more than 50% decrease in alpha content of the eIF4B protein. Biophysical characterization of the effect of eIF4B versus eIF4B‐Zn and its interactions with other (eIF) factors is also carried out. Supported by National Science Foundation grant MCB 0814051