UV resonance Raman study of streptavidin binding of biotin and 2-iminobiotin: comparison with avidin

UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin-biotin complex and new data from the avidin-2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

Elucidating the inhibitory effects of rationally designed novel hexapeptide against hen egg white lysozyme fibrillation at acidic and physiological pH

Inhibition of highly ordered cross-β-sheet-rich aggregates of misfolded amyloid proteins using rationally designed sequence-based short peptides is a promising therapeutic strategy for the treatment of neurodegenerative diseases. Here, we have explored the anti-amyloidogenic potency of a rationally designed hexapeptide (Tyr-Pro-Gln-Ile-Pro-Asn) on in vitro hen egg white lysozyme (HEWL) amyloid fibril formation at acidic pH and physiological pH using computational docking as well as various biophysical techniques such as fluorescence spectroscopy, UV–vis spectroscopy, FTIR spectroscopy, confocal microscopy and TEM. The peptide was designed based on the aggregation-prone region (APR) of HEWL and thus referred to as SqP1 (Sequence-based Peptide 1). SqP1 showed over 70% inhibition of HEWL amyloid formation at pH 2.2 and approximately 50% inhibition at pH 7.5. We propose that SqP1 binds to the APR of HEWL and interacts strongly with the Trp62/Trp63, ultimately stabilizing monomeric HEWL at both the pH conditions and preventing conformation changes in the structure of HEWL, leading to the formation of amyloidogenic fibrillar structures. A sequence-based peptide inhibitor of HEWL amyloid formation was not reported previously, making this a critical study that will further emphasize the importance of short synthetic peptides as amyloid inhibitors.     

Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

Ultraviolet-resonance raman spectroscopy of the filamentous virus Pf3: interactions of Trp 38 specific to the assembled virion subunit

The class II filamentous virus Pf3 packages a circular single-stranded DNA genome of approximately 5833 [corrected] nucleotides within a cylindrical capsid constructed from approximately 2500 [corrected] copies of a 44 residue alpha-helical subunit. The single tryptophan residue (Trp 38) of the capsid subunit is located within a basic C-terminal sequence (.R(+)WIK(+)AQFF). The local environment of Trp 38 in the native Pf3 assembly has been investigated using 229 nm excited ultraviolet-resonance Raman (UVRR) spectroscopy and fluorescence spectroscopy. Trp 38 exhibits an anomalous UVRR signature in Pf3, including structure-diagnostic Raman bands (763, 1228, 1370, and 1773 cm(-)(1)) that are greatly displaced from corresponding Raman markers observed in either detergent-disassembled Pf3, class I filamentous viruses, most globular proteins, or aqueous L-TRP. An unusual and highly quenched fluorescence spectrum is also observed for Trp 38. These distinctive UVRR and fluorescence signatures together reflect interactions of the Trp 38 side chain that are specific to the native PF3 assembly. The experimental results on PF3 and supporting spectroscopic data from other proteins of known three-dimensional structure favor a model in which pi electrons of the Trp 38 indolyl ring interact specifically with a basic side chain of the subunit C-terminal sequence. Residues Arg 37 AND Lys 40 are plausible candidates for the proposed cation-pi interaction of Trp 38. The present study suggests that raman spectroscopy may be a generally useful probe of interactions between the indolyl pi-electron system of tryptophan and electropositive groups in proteins and their assemblies.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein

The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.     

Spectroscopic analysis of acylated and deacylated myelin proteolipid protein

The effect of covalently bound fatty acid on the conformation of the myelin proteolipid protein has been studied by ultraviolet and intrinsic fluorescence spectroscopy. With dimethyl sulfoxide used as a perturbant, the exposure of Trp and Tyr residues in various mixtures of chloroform-methanol was evaluated by difference spectroscopy of the proteolipid protein (APL) and its chemically deacylated form (d-APL). The fraction of chromophoric groups exposed increased with the proportion of chloroform with 25% of the groups exposed in 1:2 chloroform-methanol and 98% in 3:1 chloroform-methanol. These conformational changes correlate well with changes in intrinsic viscosity. Values for the deacylated form were indistinguishable from those of the acylated protein, suggesting that fatty acids do not affect protein conformation in organic solvents. In water, UV difference spectroscopy indicated that the number of Tyr and Trp groups exposed in both APL and d-APL was relatively small and was independent of the molecular size of the perturbant. However, differences in the environment of the Trp groups in the two forms of the protein could be demonstrated by intrinsic fluorescence. When the protein was excited at 295 nm, the maximum emission wavelength for the acylated protein was 330 nm, whereas it was 335 nm for the deacylated form. Furthermore, the Trp groups in d-APL were more easily quenched by acrylamide than in APL, indicating that they were more exposed, or in a more hydrophilic environment, following deacylation. Protein aggregation appears to be independent of the presence of fatty acids, suggesting that the fluorescence differences between APL and d-APL are related to factors other than aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers → monomers → partially unfolded monomers → oligomeric aggregates → fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.     

Direct observation of the structural change of Tyr174 in the primary reaction of sensory rhodopsin II

Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the protein which are transmitted to a transducer protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for all the tryptophan bands within the instrumental response time, followed by a partial recovery with a time constant of 30 ps and no further changes up to 1 ns. In the 238 nm UVRR spectra, a fast recovering component was observed in addition to the 30 ps time constant component. A comparison between the spectra of the WT and Y174F mutant of SRII indicates that Tyr174 changes its structure and/or environment upon chromophore photoisomerization. These data represent the first real-time observation of the structural change of Tyr174, of which functional importance was pointed out previously.     

Identification of tyrosine residues involved in ligand recognition by the phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and UV resonance Raman studies.

Src homology 3 (SH3) domains are small noncatalytic protein modules capable of mediating protein-protein interactions. We previously demonstrated that the association of a ligand peptide RLP1 (RKLPPRPSK) causes environmental and structural changes of Trp55 and some of seven Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain by circular dichroism (CD) and 235-nm excited UV resonance Raman (UVRR) spectroscopies [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr12, Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue. Among these three residues, Tyr14 was found to be a main contributor to the UVRR spectral change upon the RLP1 binding. Interestingly, CD and UVRR analyses revealed that RLP1 associates with the Y14F and Y14H mutants in different ways. These results suggest that Tyr14 plays a crucial role in the ligand recognition, and the amino acid substitution at Tyr14 affects the mode of PI3K SH3-ligand interaction. Our findings give an insight into how SH3 domains can produce diversity and specificity to transduce signaling within cells.     

Pathway of information transmission from heme to protein upon ligand binding/dissociation in myoglobin revealed by UV resonance raman spectroscopy

Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O(2) to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O(2) or CO binding. On the other hand, binding of O(2) to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.     

Conformational changes of disulfide isomerase C induced by guanidine hydrochloride

Unfolding--refolding of Escherichia coli disulfide isomerase C (DsbC) induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbour of a single tryptophan residue Trp 140, sulfur atoms of the disulfide bond Cys 141--Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It has been revealed that, though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38 and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data), the character of parametric dependencies between fluorescence intensity recorded at 320 and 365 nm, and the existence of an isosbestic point of protein fluorescence spectra in solutions with different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation. The reversibility of this process is also shown.     

Characterization of oligomers during alpha-synuclein aggregation using intrinsic tryptophan fluorescence

The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.     

Conformational change of the dimeric DsbC molecule induced by GdnHCl. A study by intrinsic fluorescence

Unfolding-refolding of Escherichia coli DsbC, a homodimeric molecule, induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbor of the single tryptophan residue (Trp 140), the sulfur atoms of the disulfide bond Cys 141-Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It was revealed that though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38, and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data) and its parametric representation, the existence of an isosbestic point of fluorescence spectra at different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation and its reversibility.     

The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.     

The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Effect of 1-methyl-3-octyleimmidazolium chloride on the stability and activity of lysozyme: a spectroscopic and molecular dynamics studies

Herein, the binding of 1-methyl-3-octylimidazolium chloride [OMIM][Cl] ionic liquid with hen egg white lysozyme (HEWL) has been studied using fluorescence, time resolved fluorescence, UV–visible and circular dichroism (CD) spectroscopy, in combination with computational study. The fluorescence results revealed that [OMIM][Cl] quenches the fluorophore of HEWL through static quenching mechanism. The calculated thermodynamic parameters show that [OMIM][Cl] bind with HEWL through hydrophobic interactions. In addition, the negative value of Gibbs energy change (?G) indicates that the binding process was spontaneous. Furthermore, UV–vis and CD results indicate that [OMIM][Cl] induce the conformational change in HEWL and increase its enzymatic activity. Additionally, molecular docking results showed that [OMIM][Cl] binds at the active site of HEWL where both the fluorophore residues (Trp108 and Trp62) and the catalytic residues (Glu35 and Asp52) reside. Molecular dynamic simulation results show the reduction of intra-molecular hydrogen bond of HEWL when it binds with [OMIM][Cl].     

Ultraviolet resonance Raman studies reveal the environment of tryptophan and tyrosine residues in the native and partially folded states of the E colicin-binding immunity protein Im7

Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.