Photophysics of aqueous tryptophan: pH and temperature effects

Robbins, Rebecca J.; Fleming, G. R.; Beddard, Godfrey S.; Robinson, G. W.; Thistlethwaite, Peter; Woolfe, G.

doi:10.1021/ja00540a016

Cited by 218 publications

(145 citation statements)

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“…1B shows a kinetic folding trace starting with the protein fully unfolded at pH 2 (shown by CD) and then jumping to higher pH to initiate refolding at zero denaturant. A burst phase increase in fluorescence (not shown), initially attributed to the formation of a fast burst phase intermediate, is an artifact due to fluorescence quenching at the acid unfolding pH (25,26). The refolding kinetics at zero denaturant shows an initial lag phase to which standard multiexponential fitting assigns a negative amplitude (Ϫ5%).…”

mentioning

confidence: 96%

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Bédard

Mallela

Mayne

et al. 2008

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

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The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models.foldons ͉ PPOE model ͉ staphylococcal nuclease T hat proteins might fold by way of some predetermined pathway was first suggested by C. Levinthal (1), who reasoned that folding through a random search process would take far longer than the subsecond time scale often observed. Experimental work driven by this concept found evidence for distinct pathway intermediates (2). However, theoretical work showed that the energetically downhill nature of the conformational search might by itself be sufficient to drive random folding on a realistic time scale (3). No specific pathway would then be necessary. This scenario has been formalized in the funneled energy landscape view for protein folding (4-8). Following this precedent, experimental results for a number of proteins have been similarly interpreted in terms of multiple unrelated parallel pathways (9)(10)(11)(12)(13)(14)(15)(16). The critical observation is that protein folding can be kinetically heterogeneous. Different fractions of a folding population fold at different rates and exhibit different populated intermediates, suggesting alternative pathways. This view is often represented in terms of multiple tracks through the funneled energy landscape. We refer to this view as the independent unrelated pathways (IUP) model to emphasize that intermediate structures in the different tracks have no particular relationship.A much more determinate pathway is supported by structural information derived experimentally for folding intermediates in many proteins (17)(18)(19)(20). This information indicates that protein folding intermediates are definitively native-like, constructed from cooperative foldon units of the target native protein. It appears that native-like foldon units are formed and docked into place one after another in a stepwise sequence, each one guided and stabilized by pr...

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mentioning

confidence: 96%

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Bédard

Mallela

Mayne

et al. 2008

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

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“…The time evolution of the fluorescence anistropy can yield important information about the molecular motion, but the analysis of tryptophan fluorescence requires consideration of the complexities of its photophysics (15-18). Two electronic states having differently oriented transition dipoles are involved in the absorption process (19)(20)(21)(22), and frequently there were observed two fluorescent states (15,17,23,24). …”

mentioning

confidence: 99%

“…Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) but has only one tryptophan, Trp-14, and should be a simpler system with which to understand the fluorescence properties.…”

mentioning

confidence: 99%

Picosecond fluorescence decay of tryptophans in myoglobin.

Hochstrasser¹,

Negus²

1984

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

122

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The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with F6rster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.Tryptophans are useful optical probes of protein structure because of their relatively long wavelength absorption compared with polypeptides and other common amino acid residues. A first step in realizing the full potential of such probes is the development of techniques to distinguish the different tryptophans in a protein by means of their optical responses. We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1-6), 02 (1,(4)(5)(6), and NO (6, 7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.Pulsed laser techniques are needed to study tryptophan fluorescence in heme proteins as a result of very efficient energy transfer to the heme. The low fluorescence quantum yields for tryptophan in heme proteins (8) suggest that the corresponding lifetimes are in the picosecond range. Thus, to obtain direct information on the lifetimes and motional characteristics of such tryptophans it was necessary to devise experiments by which the fluorescence and its polarization could be measured with picosecond accuracy.Sperm whale (SW) Mb is known to contain the two tryptophan residues, Trp-7 and Trp-14 (9). Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) bu...

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“…Relative¯uorescence quantum yields (0 f ) were determined using L-tryptophan as standard (0 fs 0.135) [11,12]. Fluorescence quenching studies were performed by addition of ethanolic solutions of retinal or retinol acetate (3 ml, 4.37Â10 À3 M) to the respective solutions of 4 and 5.…”

Section: Fluorescence Studiesmentioning

confidence: 99%

“…It is further known that Trp-182 is located above the central polyene chain whereas Trp-189 lies below the - [6,30]. c [11,12]. d [32].…”

Section: Comparison Of the Fluorescence Of Model Chromophores With Bamentioning

confidence: 99%

Fluorescence probe properties of N-octadecamido compounds of tryptophan

Singh

Das

1998

Journal of Photochemistry and Photobiology A: Chemistry

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Two new long chain tryptophan compounds viz [3-(indolyl-3 H )-2-(N-octadecamido)]-methylpropionate (4) and sodium-[3-(indolyl-3 H )-2-(N-octadecamido)]-propionate (5) have been prepared and their¯uorescence properties (! max , 0 f , ( f , r ss , quenching) have been investigated in homogeneous media and in phosphatidyl choline vesicles. Both 4 and 5 in a membrane mimicking system of phosphatidylcholine vesicles show blue-shifted emission maxima but higher quantum yields of¯uorescence as compared to tryptophan. The steady state anisotropy values show that the molecules are immobilised in the membrane. While both the¯uorophores show single exponential uorescence decay pro®le in homogeneous media, a triple exponential¯uorescence decay is observed in vesicular medium. Thē uorescence of both the compounds are quenched by retinyl polyenes viz. retinal and retinyl acetate. The extent of quenching is more in organised media of vesicles as compared to organic solvents. A comparison of the¯uorescence data for 4 and 5 with those for retinalbinding protein bacteriorhodopsin and the apoprotein bacterioopsin reveals some similarities in the nature of interaction between retinylidene polyenes and 4 and 5, and between retinylidene Schiff base chromophore and tryptophan residues in the protein.

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Photophysics of aqueous tryptophan: pH and temperature effects

Cited by 218 publications

References 0 publications

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

Picosecond fluorescence decay of tryptophans in myoglobin.

Fluorescence probe properties of N-octadecamido compounds of tryptophan

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