Tryptophan fluorescence, intrinsic

Figure 5. Fraction unfolded FKBP measured by automated folding analysis. The experiment was performed by flow-imecting a constant 10% stream of FKBP in 9.31 M buffered urea into the second mixer (Fig. 1) and performing automated refolding using intrinsic tryptophan fluorescence (11) as described in Fig. 4.

Figures 16.10 and 16.11 present results whose aim was to deliver active kerati-nocyte growth factor (KGF) from a BP-based platform. In Fig. 16.10, we illustrate the measurement/screening strategy based on the intrinsic tryptophan fluorescence from KGF Fig. 16.10a shows the KGF emission spectral (kex = 257 nm) shift between native and denatured KGF. Figure 16.10b shows that there is a strong correlation between the observed KGF emission maximum and the KGF activity in vitro. Figure 16.11 illustrates the effect of BP polymers (A), PLA/PGA ratio (B), concentration of surfactant aerosol-OT (AOT) (C), molar ratio of water to AOT,...

Figure 4.13 pH-Titration of protein Hsp47 monitored by intrinsic tryptophan fluorescence spectroscopy (excitation 295 nm). Data is also indicative of a two stage transition between an Alkali stable state and an Acid stable state via a transient intermediate state (see Fig. 4.8). Fluorescence spectroscopy also reveals that Hsp47 will undergo reversible pFI-driven trans-conformational changes (see Chapter 7) (Reproduced from Momma et al., 2008). /em(iC) is fluorescence emission intensity.

Aside from this application, intrinsic tryptophan fluorescence may be used in combination with fluorescence quenching agents to discriminate between those tryptophan residues more surface accessible or solvent exposed than others. This relies upon the Stern-Volmer equation... 

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

Casimiro, D. R., Wong, L. L., Colon, J. L., Zewert, T. E., Richards, J. H., Chang, I-J., Winkler, J. R. and Gray, H. B, 1993, Electron transfer in ruthenium / zinc porphyrin derivatives of recombinant human myoglobins. Analysis of tunneling pathways in myoglobin and cytochrome c. Journal of American Chemical Society 115, 1485-1489. Chang, Y-C. and Ludescher, R. D, 1994, Local conformation of rabbit skeletal myosin rod filaments probed by intrinsic tryptophan fluorescence. Biochemistry 33, 2313 -2321. 

The affinity of 2 — P for extracellular Na" " appears to depend on the presence or absence of extracellular [46]. In the absence of extracellular a site of high affinity for Na ( d=0.5 mM) is exposed through which Na" inhibits the spontaneous hydrolysis of 2 — P [46,47] or its reformation following hydrolysis [13]. Increasing the external Na concentration abolishes this inhibition and increases the D for Na to about 30 mM [9]. The , P 2 — P transition, like the primary phosphorylation, is reversible. Increasing the Na concentration raises the fraction of ADP-sensitive phosphointermediate [40] and decreases the intrinsic tryptophan fluorescence evoked by phosphorylation with ATP to 2 — P [48]. 

The concepts described in the preceding sections allow us to readily understand the characteristics of newly devel oped probes. One recent example is the all-/ra/w isomer of 8,10,12,14,16-octadecapentaenoic acid (r-COPA, Figure 11.30). This probe is essentially insoluble and/or nonfluo rescent in water, so that the only emission is from /-COPA bound to membranes. The absorption spectrum of t COPA is centered at 330 nm, making it an effective acceptor for the intrinsic tryptophan fluorescence of membrane-bound proteins. The effectiveness of /-COPA... 

The intrinsic tryptophan fluorescence emission spectra of Sac7 and Sso7 (5 p, Af protein in 10 mM KH2PO4, pH 6.8) are obtained with excitation at 295 nm using 4 nm excitation and emission slit widths. Excitation at 295 nm prevents contributions from the two tyrosine and two phenylalanine residues. An emission maximum at 350 nm is similar to that of free tryptophan and indicates significant solvent exposure, consistent with the NMR solution structure. Addition of double-stranded DNA [e.g., duplex poly[d(GC)]] leads to quenching of tryptophan fluorescence by nearly 90%. A blue shift of the emission maximum to 340 nm is also observed. 

DNA binding constants and site sizes are obtained from fluorescence titration data by nonlinear regression. The observed intrinsic tryptophan fluorescence quenching, Qobs, is defined by... 

Gorinstein, S. Goshev, I. Moncheva, S. Zemser, M. Weisz, M. Caspi, A. Libman, I. Lerner, H. T Trakhtenberg, S. Martin-BeUoso, O. Intrinsic tryptophan fluorescence of human serum proteins and related conformational changes. J. Protein Chem., 2000, 79(8), 637-642. 

In contrast to thermally induced aggregates, isolated aggregates of bevacizumab produced by up to 30 freeze-thaw cycles showed little change in HX kinetics [30]. In this case, thCTe was also no appreciable change in intrinsic tryptophan fluorescence or ANS fluorescence between the freeze-thaw-induced aggregate and the control mAb. Results from HX-MS analysis in this study correlated well with fluorescence measurements, and the fluorescence results were consistent with observations in a complementary freeze-thaw and thermally induced aggregate study with another mAb [57]. 

Other data, relevant to a nearby residue, Trp-153, also support the notion that the nicotinamide moiety of bound NAD may be in contact with this region of the primary structure of the toxin. Binding of NAD to fragment A strongly quenches the intrinsic tryptophan fluorescence of the fragment and generates a weak absorbance peak (X ax ) [ 1- Both phenomena depend upon an N-substituted nicotinamide... 

This particular reaction has been chosen for the reason of its high value of standard chemical affinity for this reaction (j / = — 7.1 kcal/mole). As we noted above, due to this circumstance the system behavior can reveal the deviation from that prescribed by the classical Arrhenius mechanism. The conformational changes in malatdehydrogenase were tested by measuring the average life-time of intrinsic tryptophane fluorescence (ff). This parameter is known to be sensitive to the immediate surrounding of tryptophane residues. The chemical transformation of the substrate was detected from changes in the coenzyme redox state measured in terms of the sample optical density at 340 nm (NADH absorption maximum). 

Bell J, El Meskini R, DAmato D, Mains RE, Eipper BA. 2003. Mechanistic investigation of peptidylglycine a-hydroxylating monooxygenase via intrinsic tryptophan fluorescence and mutagenesis. Biochemistry 42 7133-7142. 

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