Alteration of tryptophan fluorescence properties upon dissociation of Lumbricus terrestris hemoglobin (original) (raw)
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Environment of tryptophan side chains in proteins
2000
Abstract Although relatively rare, the tryptophan residue (Trp), with its large hydrophobic surface, has a unique role in the folded structure and the binding site of many proteins, and its fluorescence properties make it very useful in studying the structures and dynamics of protein molecules in solution. An analysis has been made of its environment and the geometry of its interaction with neighbors using 719 Trp residues in 180 different protein structures.
Biophysical Chemistry, 2002
The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C and C atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from thé 2 d2g C. Bosch Cabral et al. / Biophysical Chemistry 97 (2002) [139][140][141][142][143][144][145][146][147][148][149][150][151][152][153][154][155][156][157] of 4.6 and 2.1 ns, which contribute approximately 90% of the total fluorescence, one of 300 ps (4%), and the last one of 33 ps (7.4%). It is clear that the equilibrium structure resulting from homology modeling explains the subnanosecond fluorescence lifetimes, while the nanosecond range lifetimes require more information about the protein in solution, since there is a significant contribution of lifetimes that resemble the apo molecule. ᮊ
Fluorescence Spectroscopy of the Tryptophan Microenvironment in Carcinus aestuarii Hemocyanin
Zeitschrift für Naturforschung C, 2002
The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.
Biophysical Journal, 1997
The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membranebound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the a-amino group of TOE occurs with an apparent PKa of -7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.
A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin
Biophysical Chemistry, 1983
Judohirrnt has a wry short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components. one of which (component A) corresponds tc the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid cnvironmrnt (like the single trypcophan residue in azurin), !he other (component I) being due to the emission of buried tryptophnn residues Iocnwd in 3 rather polar environment. Treatment of bacteriorhodopsin by NaBH,. fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A. an increase in that of component I and the appenrtlncc of spectral components corresponding to the emission oi surface tryptophan residues. Temperature inducts at least two distinct changes c: the fluorescence parameters of the protein: one change occurs from 45 to 65OC. the other irom 65 to 9OOC. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline drnaturation of bacteriorhodopsin registered by tryptophnr. fluorescence begins at pH > 11.0. 03014622/83/.503.00 0 !283 Elsevier Science Publishers B.V.
Protein Science, 2007
Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.