ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 318 (2003) 300–308 www.elsevier.com/locate/yabio
Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin Oktay K. Gasymov, Adil R. Abduragimov, Taleh N. Yusifov, and Ben J. Glasgow* Department of Pathology and Department of Ophthalmology, UCLA School of Medicine, Los Angeles, CA 90095, USA Received 4 February 2003
Abstract Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1 La and the 1 Lb electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1 Lb spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1 Lb component from the rest of the 1 Lb was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1 La and 1 Lb components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1 Lb component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra. Ó 2003 Elsevier Science (USA). All rights reserved.
Recently, a great deal of information about protein structure has been gained from spectroscopic studies of tryptophan [1–7]. The secondary structure of an entire protein, tear lipocalin, has been resolved by site-directed tryptophan fluorescence [7]. However, fluorescence emission mainly originates from the 1 La electronic level whereas CD spectroscopy features both the 1 La and 1 Lb electronic transitions. From near-UV CD, information is potentially attainable from the kmax of the spectrum, and from the sign and relative intensities of the tryptophan spectral components. Near-UV CD spectra of tryptophan in proteins are highly sensitive to interactions between nearby groups. A variety of mechanisms may create an asymmetric environment and induce CD bands [8]. The wavelength position and sharpness of aromatic CD bands in proteins are often strongly influenced by the environment of each side chain, e.g., hydrogen bonding, polar or charged groups, and polarizability. Therefore, near-UV CD spectra of tryptophan potentially hold valuable conformational information about proteins. Extracting this information * Corresponding author. Fax: 1-310-794-2144. E-mail address:
[email protected] (B.J. Glasgow).
is predicated on the resolution of the CD spectra of tryptophan and has been approached by fitting spectra as the sum of the 1 La and 1 Lb components [9]. However, the published methods for fitting tryptophan spectra do not work well for certain tryptophan spectra and therefore cannot be easily applied as a general procedure. The focus of this paper is to improve the resolution of the tryptophan components by a broadly applicable fitting algorithm. Experimental and theoretical considerations indicate that when the tryptophanyl side chain is less than 10 A from another aromatic side chain (His, Tyr, Trp, Phe), the near-UV CD may be especially intense due to l–l coupling [8]. Coupling between the near-UV transition of tryptophanyl side chain and the p–p transitions of may also give apprepeptide bonds within about 8 A ciable CD intensity [8]. The near-UV tryptophan absorption band is composed of two distinctive electronic transitions, 1 La and 1 Lb , whose transition moments are approximately orthogonal [10]. The dipole strength of the 1 La transition is much larger than that of the 1 Lb transition. The 1 La absorption band does not show any obvious vibrational structure [8–10]. The maximum of 1 La band is about
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O.K. Gasymov et al. / Analytical Biochemistry 318 (2003) 300–308
279 nm for Trp in buffer [9,10]. The 1 La band is highly sensitive to the environment; it is influenced by polarity, polarizability, and the formation of hydrogen bonds with the indole NH group. Depending on the position and sign of a charged group relative to indole group, the 1 La band can be shifted to the red or blue [11]. The 1 Lb band is characterized by two prominent vibronic bands, 0-0 and 0 + 850 cm1 . A third vibronic band is poorly resolved and mainly appears as a shoulder of the spectrum. The 1 Lb is much less sensitive to the environment than the 1 La . Changes in polarizability of the surroundings may shift the tryptophanyl 1 Lb band by only a few nanometers. The 0-0 1 Lb band in proteins lies between 287 and 293 nm [8]. Near-UV CD tryptophanyl spectra in model compounds and in proteins have been classified into four types [8]: (1) spectra with a strong 1 Lb band but with little or no obvious intensity of the 1 La band, (2) spectra in which the 1 La electronic transition dominates, (3) spectra in which both 1 La and 1 Lb electronic transitions have obvious CD bands, and (4) spectra that feature a large mismatch between the wavelengths of the CD bands and those of the absorption bands, presumably due to significantly different CD from multiple rotamers of Trp. Some success has been achieved in resolving the 1 La and the 1 Lb components either by analyzing the fluorescence excitation spectra, by analyzing the polarization of absorbance, or by deconvolving spectra obtained for a Trp derivative to resolve the Trp absorbance spectra [9,10]. Two basic forms of 1 Lb tryptophanyl spectra have emerged for fitting CD spectra, that of Barth et al. [9] and that of Valeur and Weber [10]. The relative amplitude of the 0-0 transition compared to the rest of the 1 Lb spectrum is much different in these two forms. The variations of relative intensities of 0-0 transitions were suggested to be the result of different relative amplitudes of vibronic transitions of Trp in buffer versus Trp included in a protein structure [9]. Here, we separated the 1 Lb (0-0) transition from the rest of the 1 Lb spectrum to allow variation of these parameters independently to improve fitting in mutants exhibiting the first three types of tryptophanyl CD spectra. These fitting results are compared to those obtained using the 1 Lb tryptophan forms and the method as described, which did not include independent variation of the 1 Lb (0-0) transition [9]. The fourth type of tryptophanyl CD spectra, the multicomponent type, requires a second 1 La band and will be the subject of future studies.
Materials and methods
301
to clone the TL gene spanning bases 115–592 of the previous published sequence [13] into pET 20b (Novagene). We prepared a TL mutant, W17Y, as a template to construct mutants in which a single tryptophan was substituted for other amino acids. Mutants for tear lipocalin that have different combinations of the 1 La and 1 Lb bands with regard to the intensity and sign were chosen. These mutants included tryptophan CD spectra that featured low intensity of both 1 La and 1 Lb bands (W17Y/D94W), a dominant 1 La band (W17Y/R26W), a dominant 1 Lb band (W17Y/Y99W), prominent 1 La and 1 Lb bands with the same sign (W17Y/I97W), and 1 La and 1 Lb bands of opposite signs (W17Y/I98W and W17Y/G103W) [7]. The mutant plasmids were transformed in Escherichia coli BL21-(DE3). Cells were cultured and proteins were expressed and purified as previously described [7]. The protein concentration was determined by the biuret method [14]. Absorption spectroscopy UV absorption spectra were recorded at room temperature using a Shimadzu UV-2400 PC spectrophotometer. CD spectral measurements Spectra were recorded (Jasco 810 spectropolarimeter, 10-mm path lengths for near-UV spectra) using a protein concentration of 1.2 mg/mL. Sixteen scans from 250 to 320 nm were averaged. Results were recorded in millidegrees and converted to molar De in M1 cm1 . Fitting of the CD spectra CD spectra were first analyzed using the 1 Lb tryptophanyl spectra of Barth et al. [9] and Valeur and Weber [10]. To derive both forms, the 0-0 band of 1 Lb was separated from the rest of the Trp 1 Lb spectrum (1 Lb (r)) and intensities of these parameters were allowed to vary independently. This procedure allowed us to fit near-UV CD spectra of Trp residues with the same precision with either form of 1 Lb . While this method was adequate, fitting was improved by deconvolving the 1 Lb band of Barth et al. into five Gaussian components to fit the absorption spectrum of N-acetyl-L -tryptophan amide (NATA)1 in buffer. No deconvolution was done for the 1 La band. For the purpose of fitting CD spectra, the Gaussian component corresponding to the 0-0 transition was manipulated separately from the remaining four Gaussian components that were manipulated as a sum.
Site-directed mutagenesis and plasmid construction The TL cDNA in PCR II (Invitrogen, San Diego, CA), previously synthesized [12], was used as a template
1 Abbreviations used: NATA, N-acetyl-L -tryptophan amide; TL, tear lipocalin.
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Programs For deconvolution of Gaussian components, Microcal ORIGIN software (Northampton, MA) was used. Manipulation and fitting of tryptophan absorption and CD spectra were carried out using computer programs written in LabVIEW (National Instruments, Austin, TX). The computer was programmed to find the best linear combination of 1 Lb (0-0) transition, 1 Lb (r), 1 La band, the remaining aromatic component (designated as the W17Y contribution), and a baseline component permitting the shift of the whole spectrum. The aromatic composition of the W17Y mutant includes six tyrosine and three phenylalanine residues. However, since the CD contribution from phenylalanine occurs below 270 nm and there is no obvious fine structure in this region, the remaining aromatic component originates from the tyrosine residues. However, in one mutant, W17Y/ Y97W, there are only five tyrosines. Previously, we demonstrated that the near-UV CD of Y97C was minimally changed from TL, indicating that the tyrosine 97 has a very low CD contribution [15]. Insertion of tryptophan close to an existing tyrosyl side chain may alter the conformation around a Tyr side chain and the CD contribution. An allowance of 10% variation in the contribution of W17Y permitted fitting of the spectra in all of our mutants, providing further evidence that the tyrosine contribution was minimally altered by the insertion of trytophan residues. In all spectra, 250 points were generated in the range 260–320 nm with an increment of 0.2 nm. The 1 La and 1 Lb bands were shifted in steps of 0.2 nm. The 1 Lb components, 1 Lb (0-0) transition and 1 Lb (r), were in general shifted as a group, but independent shifts within a range of 1 nm were also allowed. Because of uncertainty in determination of protein concentrations, the intensity of the tyrosyl component was permitted to vary 10% with either a positive or a negative sign. A minimum v2 (the sum of the square of the deviations between the fit and the corresponding experimental spectrum) value was chosen for the best fit: X v2 ¼ ðfi yi Þ2 :
support plane analysis was used [3]. After finding the parameters that correspond to the minimum v2 value, fitting was reperformed, changing the value of one parameter and keeping it constant while allowing variation in the other component values to minimize v2 to a new value. Because the intensities of the CD signals of single Trp mutants vary over a large range it was convenient to represent the variation of amplitude as I/Imin , where Imin is the amplitude when v2 is at a minimum. For determination of the range of parameter values consistent with the data we have chosen the probability (P) value of 0.32, such that when the value of P is less than 0.32, there is a 68% chance that the parameter value is consistent with the data (definition of standard deviation). Detailed analysis and parameters for the F statistics have been compiled [3]. Using this analysis [3] Fv was estimated to be 1.024.
Results and discussion NATA Initially, we attempted to use the 1 Lb tryptophanyl spectra of Barth et al. [9] and Valeur and Weber [10] to fit CD spectra of lipocalin mutants. However, a close fit was not obtained in certain instances, e.g., W17Y/I98W. Fitting could be further improved by deconvolving the 1 Lb spectrum into the 1 Lb (0-0) transition and four additional Gaussian components. There is direct experimental evidence for these five components in studies of a related model compound, 5-methoxyindole [16]. The deconvoluted spectrum of NATA with the derived 1 Lb band, 1 La band, and the fitting curve is shown in Fig. 1. By varying the amplitude of the derived spectrum for the 1 Lb (0-0) transition independently from the sum of the
i
fi is the sequence of fitted values and yi is the sequence of observed values. If variation in one parameter minimized the v2 value, other parameters were varied on either side of the previous minimum values to find a new minimum. To better understand the accuracy of the parameters, the F statistic, the ratio of v2 /v2min was determined. In general, this ratio depends on the degrees of freedom (m) for each fit, m ¼ n p, which is constant for the assessed spectra, where n is the number of data points and p is the number of floating parameters. For an estimation of the confidence interval in the recovered parameters, a
Fig. 1. Deconvolution of the spectral components of NATA to derive the best fitting procedure for near-UV CD spectra of tryptophan. Five Gaussian components were necessary to provide the best fit to the spectrum.
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Fig. 2. Derivation of the 1 Lb band type of Barth et al. (A) and that of Valeur and Weber (D) from the 1 Lb band deconvolved from the absorption spectrum of NATA (B and C). To achieve the spectrum in D, the intensity of the 1 Lb (0-0) transition component was increased 1.7-fold and the spectrum was normalized to unity.
remaining components (1 Lb (r)), the 1 Lb spectra of Barth et al. [9] and Valeur and Weber [10] could be developed (Fig. 2). Fitting of the spectra from lipocalin mutants was undertaken using the derived 1 Lb (0-0) transition and 1 Lb (r) components of the Trp 1 Lb spectrum. Lipocalin mutants Analyses of different Trp mutants of TL show that the amplitude of the 0-0 band of 1 Lb varies with location of the Trp residue in the protein. The deconvolution of the different tryptophan mutants of tear lipocalin is shown in Figs. 3–8 and the fitting parameters are shown
Fig. 3. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ K94W. (A) Observed and best fit spectra. (B) Deconvolved components.
in Table 1. It is evident from these data that the tryptophans represented in these mutants have vastly different near-UV CD 1 Lb characteristics that are representative of the different types of tryptophan spectra. W17Y/F99W, shows a higher intensity of the 1 Lb bands compared to the 1 La bands (CD type 1). The mutant, W17Y/R26W shows a high intensity of the CD signal but lacks fine structure (CD type 2). W17Y/I98W, shows a strongly positive 1 La band and strongly negative 1 Lb band (CD type 3). In each case a good fit was obtained for the observed spectrum. While it is premature to draw firm conclusions, the newly resolved
Fig. 4. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ Y97W. (A) Observed and best fit spectra. (B) Deconvolved components.
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Fig. 5. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ I98W. (A) Observed and best fit spectra. (B) Deconvolved components.
Fig. 7. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ G103W. (A) Observed and best fit spectra. (B) Deconvolved components.
Fig. 6. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ F99W. (A) Observed and best fit spectra. (B) Deconvolved components.
components permit some speculative comparisons to known structural information. The CD spectrum of W17Y/D94W shows a lowintensity Trp CD signal, indicating a symmetric environment consistent with the known position in the
Fig. 8. Fitting of near-UV CD spectrum of tryptophan mutant W17Y/ R26W. (A) Observed and best fit spectra. (B) Deconvolved components.
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Table 1 Parameters of deconvolution of Trp spectra in single Trp mutant proteins of TL Mutant
1
1
La
1
Lb
1
W17Y/K94W W17Y/Y97W W17Y/I98W W17Y/F99W W17Y/G103W W17Y/R26W
kmax (nm)
De of the maximum (M1 cm1 )
kmax of (0-0 band) (nm)
De of the maximum (M1 cm1 )
283.7 285.2 273.4 278.3 278.9 278.0
)0.18 +1.36 +10.02 +0.54 )0.70 +1.84
291.5 288.4 290.8 291.3 292.3 289.7
)1.18 +1.58 (+2.47) )2.80 ()3.63) +2.07 (+2.07) +0.60 (+0.67) )0.26 ()0.27)
(287.2) (290.2) (291.0) (292.0) (289.7)
Lbð0-0Þ / Lbð0 þ 850Þ
0.16a 0.43 (0.67) 0.53 (0.68) 0.79 (0.87) 0.98 (1.09) 1.4a
1
La /1 Lb
+3.2a +0.4 )3.6 +0.3 )1.1 )7.0a
W17Y contribution
v2
1.00 1.10 0.90 0.92 1.06 0.90
0.015 0.055 0.058 0.030 0.046 0.016
Values in parentheses are apparent values. a Value of low precision.
loop F–G (Fig. 3). Previously, Trp at position 94 was shown to have a highly red-shifted fluorescence emission with kmax of 349.3 nm, indicative of solvent exposure [2]. The amino acids at positions 97–99 and 103 are on the b strand G of the lipocalin barrel and feature different environments [2,7]. In particular, the side chain mobility and environment of positions 98 and 99 were extensively studied by fluorescence and EPR spectroscopies [2,15], warranting comparison to the deconvolved CD spectra. Trp at position 99 shows a blue-shifted fluorescent emission with kmax of 325 nm and the most buried position in b strand G of the lipocalin barrel and is immersed in a hydrophobic environment [2]. EPR demonstrated that the nitroxide side chain at 99 was the most motionally restricted in b strand G [15]. The nearUV CD spectra of Trp99 is dominated by the positive 1 Lb component spectrum. A highly resolved 1 Lb (0-0) component at 291.3 nm is red-shifted compared to position 97, consistent with a more hydrophobic environment (Figs. 4 and 6, Table 1). Unfortunately, we could not resolve the wavelength maximum of the 1 La band of W99 precisely, because it has a low intensity (Figs. 6 and 10). The side chain of I98 is directed toward the outside of the lipocalin barrel. Trp98 shows a relatively redshifted emission, kmax of 329 nm, compared to Trp99, although this value is still considered mainly hydrophobic [2,17]. In contrast to Trp99, Trp98 features strong 1 La (positive) and 1 Lb (negative) components (Fig. 5). The kmax 1 La CD band is blue-shifted to 273.4 nm and has a high intensity. The induced Trp CD may be the result of interactions with side chains at depending on the orientation and nadistances <15 A ture of the side chain. Some of these interactions may involve tertiary contact (nearest neighbor interactions) [15,18]. Unlike position 99, the nitroxide-labeled side chain at position 98 showed two populations by EPR. These populations differ in mobility, indicative of tertiary interactions [15]. However, the induced CD observed for 98 is not necessarily the result of the tertiary
interaction observed by EPR. Alternatively, there are aromatic residues in proximity of 98 but outside the range for tertiary contact that could produce CD signal. RET measurements combined with homology modeling have demonstrated that at least three tyrosyl groups (introduced Tyr17, Tyr97, and Tyr100), and His96 and of Trp98 [7,19]. It is Phe99 residues are within 15 A known that Tyr, His, and Phe side chains may induce high CD intensity, such as that seen for Trp98. One of the difficulties in fitting the tryptophan spectra with previous methods has been handling the case where the relative amplitude of the 1 Lb 0-0 transition band is low. This is evident from comparison of the spectra of Trp98 and Trp99 in Figs. 5 and 6, respectively. The relative amplitude of the 0-0 transition of Trp98 is lower than that for Trp99. When we attempted to fit the spectrum of Trp98 with the 1 Lb of Barth et al. [9], we obtained poor fitting. However, when independent variation of the 1 Lb components was permitted, a good fit was obtained (Fig. 5). In contrast for Trp99 with a relatively high amplitiude of the 0-0 transition, a good fit was obtained using either the 1 Lb type of Barth et al. [9] or that derived from our method. The possible mechanisms for relatively low amplitude of the 0-0 transition include side chain heterogeneity associated with varying positions of the 1 Lb and perhaps interactions with other residues [20,21]. The CD spectrum of Trp97 (W17Y/Y97W) is composed of a strong 1 Lb (positive) and a relatively weak 1 La (positive) component (Fig. 4). The striking feature of the CD spectrum of Trp97 is the red-shifted 1 La band with kmax of 285.2 nm and the blue-shifted 1 Lb band (kmax of 0-0 transition is 288.4 nm). The relative amplitude of the (0-0) component of Trp97 1 Lb CD spectrum resembles that of Trp98. Available structural information may explain the CD findings for position 97. By EPR two populations, differing in mobility, were identified for the nitroxide side chain at position 97, similar to that of 98. The emission maximum of Trp97 is about 334.4 nm and is consistent with a more polar environ-
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ment than for position 98 [2]. Data from fluorescence and modeling experiments show that amino acid 97 is part of b strand G with its side chain located at the tip of closed end of the lipocalin barrel [2,7]. This side chain is in close proximity to the charged amino acid, R118, a residue that is highly conserved in the lipocalin family [22]. The mutation of Tyr to Trp at position 97 results in a modeled position for the benzene ring in close proximity to the þ NH2 group of the side chain on R118. This finding is in accord with the published observation that a positive charge in close proximity to the benzene ring of Trp induces a red shift of the 1 La band [11]. The resulting polar environment also accounts for the observed blue shift for the 1 Lb band [3]. The CD spectrum of Trp103 (W17Y/G103W) shows a negative 1 La band and a positive 1 Lb band of similar amplitudes. The Trp CD intensity is less at position 103 than at positions 97–99. This finding concurs with the model of TL that positions the side chain of Trp103 pointing toward the lipocalin barrel, but located at a shallow position compared to that of 99. EPR studies confirm that the nitroxide-labeled side chain at position 103 has more mobility than that at positions 97–99, but two populations for nitroxide side chain at 103 are not as apparent as those for 97 and 98. Interestingly, the 1 Lb (0-0) band of Trp103 CD has greater resolution (Fig. 7, Table 1) and the value of parameter 1 Lb (0-0)/1 Lb (0 + 850) for Trp103 is closer to that of 99 than that of Trp97 and Trp98. It is intriguing to speculate that the resolution of the 1 Lb (0-0) band may be indicative of the lack of heterogeneity of tertiary interactions. Conversely the relatively poor resolution of the components seen with Trp97 and Trp98 may reflect a shift of the 0-0 transition from heterogenous tertiary interactions. Further work is needed to confirm these speculations. The CD spectrum of Trp26 (W17Y/R26W) is clearly different from the rest of the CD spectra (Fig. 8). The near-UV CD spectrum of Trp26 is dominated by the positive 1 La . The contribution from the 1 Lb component is very low, resulting in poor resolution (Figs. 9 and 10). Trp26 is located at the N-terminal end of the longest loop A-B [7]. The emission maximum of Trp26, 342.5 nm, is consistent with its location at the protein surface but is not as mobile as Trp94 [2,7]. Error analysis Ratios of v=vmin for some parameters are shown in Figs. 9 and 10. The precision of fitting is reflected in the shape of the asymmetric parabola. A sharply curving parabola indicates that the error fits within a narrow range. It is evident from Fig. 9 that the precision of fitting of k max for 1 La band and 1 Lb (0-0) band are best in mutants that exhibit a high amplitude and/or fine structure in CD. In general the precision is low for the 1 La band (e.g., W17Y/F99W, W17Y/Y97W, and W17Y/
Fig. 9. Error analysis of kmax of 1 La and 1 Lb 0-0 transition.
G103) unless there is a high intensity (e.g., W17Y/R26W and W17Y/I98W). Least precise are mutants that have low intensity and lack structure (e.g., W17Y/D94W). Because the 1 Lb (0-0) band exhibits fine structure the precision is higher than that of 1 La band even with lower intensity. The accuracy of the determined amplitudes of the components, 1 La , 1 Lb (0-0), and 1 Lb (r) are shown in Fig. 10. In some cases (e.g., R26 and D94) there is greater uncertainty of the 1 Lb components where their contributions are low. An empirical best-fit method is provided to resolve the 1 La and 1 Lb components of CD spectra of Trp in different protein environments. This method permits better fitting of the tryptophan spectra, particularly when the 1 Lb 0-0 transition shows relatively low intensity. For the mutants featured in this paper, fitting was accomplished with a single 1 La band. However, in some
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Acknowledgments We thank Dr. Joseph Horwitz for access to the CD instrument and Christian Altenbach for his guidance in LabVIEW programming. This study was supported by USPHS NIH EY11224 and EY00331.
References
Fig. 10. Error analysis of intensity of individual components 1 La , 1 Lb (0-0), and 1 Lb (r).
cases a second 1 La band is required for fitting, presumably because of separate and distinct 1 La bands produced by multiple populations of tryptophan rotamers. Tryptophan near-UV CD spectra that require two 1 La bands will be the subject of future work. Although the exact mechanisms of induced CD in the descriptions above are speculative, the resolution of the tryptophan components presents the possibility of extending this analysis to the conformation of proteins as more information is gained for single Trp CD spectra of a variety of proteins.
[1] K.T. OÕNeil, H.R. Wolfe Jr., S. Erickson-Viitanen, W.F. DeGrado, Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity, Science 236 (1987) 1454–1456. [2] O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov, B.J. Glasgow, Solution structure by site directed tryptophan fluorescence in tear lipocalin, Biochem. Biophys. Res. Commun. 239 (1997) 191–196. [3] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Plenum Press, New York, 1999. [4] Y. Chen, M.D. Barkley, Toward understanding tryptophan fluorescence in proteins, Biochemistry 37 (1998) 9976–9982. [5] M. Kintrup, P. Schubert, M. Kunz, M. Chabbert, P. Alberti, E. Bombarda, S. Schneider, W. Hillen, Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding, Eur. J. Biochem. 267 (2000) 821–829. [6] B. Liu, R.K. Thalji, P.D. Adams, F.R. Fronczek, M.L. McLaughlin, M.D. Barkley, Fluorescence of cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid: a single tryptophan chi(1) rotamer model, J. Am. Chem. Soc. 124 (2002) 13329–13338. [7] O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov, B.J. Glasgow, Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for freatures that confer promiscuity in ligand binding, Biochemistry 40 (2001) 14757–14762. [8] E.H. Strickland, Aromatic contributions to circular dichroism spectra of proteins, CRC Crit. Rev. Biochem. 2 (1974) 113–175. [9] A. Barth, S.R. Martin, P.M. Bayley, Resolution of Trp near-UV CD spectra of calmodulin-domain peptide complexes into the 1 La and 1 Lb component spectra, Biopolymers 45 (1998) 493–501. [10] B. Valeur, G. Weber, Resolution of the fluorescence excitation spectrum of indole into the 1 La and 1 Lb excitation bands, Photochem. Photobiol. 25 (1977) 441–444. [11] P.R. Callis, B.K. Burgess, Tryptophan fluorescence shift in proteins from hybrid simulations: an electrostatic approach, J. Phys. Chem. B 101 (1997) 9429–9432. [12] B.J. Glasgow, C. Heinzmann, T. Kojis, R.S. Sparkes, T. Mohandas, J.B. Bateman, Assignment of tear lipocalin gene to human chromosome 9q34-9qter, Curr. Eye Res. 11 (1993) 1019–1023. [13] B. Redl, P. Holzfeind, F. Lottspeich, cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein super family, J. Biol. Chem. 267 (1992) 20282–20287. [14] D. Bozimowski, J.D. Artiss, B. Zak, The variable reagent blank: protein determination as a model, J. Clin. Chem. Clin. Biochem. 23 (1985) 683–689. [15] B.J. Glasgow, O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov, C. Altenbach, W.L. Hubbell, Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling, Biochemistry 38 (1999) 13707–13716. [16] E.H. Strickland, C. Billups, Oscillator strengths of the 1La and 1Lb absorption bands of tryptophan and several other indoles, Biopolymers 12 (1973) 1989–1995. [17] O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov, B.J. Glasgow, Resolution of ligand positions by site directed tryptophan fluorescence in tear lipocalin, Protein Sci. 9 (2000) 325–331.
308
O.K. Gasymov et al. / Analytical Biochemistry 318 (2003) 300–308
[18] W.L. Hubbell, A. Gross, R. Langen, M.A. Lietzow, Recent advances in site-directed spin labeling of proteins, Curr. Opin. Struct. Biol. 8 (1998) 649–656. [19] O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov, B.J. Glasgow, RET and anisotropy measurements establish the proximity of the conserved Trp17 to Ile98 and Phe99 of tear lipocalin, Biochemistry 41 (2002) 8837–8848. [20] E.H. Strickland, C. Billups, E. Kay, Effects of hydrogen bonding and solvents upon the tryptophanyl 1 La absorption band.
Studies using 2,3-dimethylindole, Biochemistry 11 (1972) 3657– 3662. [21] E.H. Strickland, J. Horwitz, E. Kay, L.M. Shannon, M. Wilchek, C. Billups, Near-ultraviolet absorption bands of tryptophan. Studies using horseradish peroxidase isoenzymes, bovine and horse heart cytochrome c, and N-stearyl-L -tryptophan n-hexyl ester, Biochemistry 10 (1971) 2631–2638. kerstrom, D.R. Flower, J.-P. Salier, Lipocalin: unity in [22] B. A diversity, Biochim. Biophys. Acta 1482 (2000) 1–8.