1 Laboratory of Experimental and Computational Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-5677, USA, 2 Institute of Macromolecular Compounds, Russian Academy of Sciences, 199004 St Petersburg and 4 Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia
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Abstract |
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Keywords: apomyoglobin/direct energy transfer/fluorescence/molten globule
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Introduction |
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The revealing of equilibrium and kinetic intermediates in protein folding (see Ptitsyn, 1995, for references) has confirmed that the equilibrium intermediate shares a number of structural features with the kinetic intermediate (Baldwin, 1993
) and, thus, can be considered as its static model. The study of the equilibrium intermediate is of fundamental interest itself since the molten globule might exist in a living cell and participate in important physiological processes (Bychkova and Ptitsyn, 1993
). Moreover, it was predicted (Bychkova and Ptitsyn, 1995
) and confirmed experimentally (Zhang and Peng, 1996
) that a number of genetic diseases can be caused by mutations that stop protein folding at the molten globule stage.
The first detailed analysis of the structural similarity of the molten and the native globules was achieved by a combination of hydrogen exchange with 2D NMR techniques. It was established that some -helixes in cytochrome c (Roder et al., 1988
; Jeng et al., 1990
),
-lactalbumin (Baum et al., 1989
), apomyoglobin (Hughson et al., 1990
; Jennings and Wright, 1990
), lysozyme (Redford et al., 1992
) and other proteins form at earlier stages of the protein folding and are relatively stable in the equilibrium molten globule state. Since these helixes form clusters in the native state of the corresponding proteins, it was suggested that these clusters might be formed also in equilibrium and kinetic intermediates. However, this assumption never has been supported by direct experimental data and nothing is known about the structure of these possible complexes.
Very little progress has been made so far in the experimental investigation of the 3D structural similarity of native and molten globule states. The molten globule cannot be crystallized and its NMR spectrum shows very broad resonance and a small dispersion of chemical shifts. Ewbank and Creighton (1991) proposed testing the similarity of these structures by the restoration of SS bonds in a reduced molten globule state. They found that SS bonds are formed randomly in the molten globule state of human -lactalbumin and concluded that the molten globule state in
-lactalbumin is much closer to an unfolded, but collapsed, form than to an expanding native conformation. Peng and Kim (1994), however, showed that only native SS bonds are formed by four SH groups of the single
-helical subdomain of this protein in the molten globule state. On the other hand, the same group found a random formation of SS bonds in the molten globule state of intact
-lactalbumin containing both
-helical and ß-structural subdomains (Wu et al., 1995
). Finally, these authors concluded that in the molten globule state of
-lactalbumin its helical subdomain has a native-like folding pattern whereas its ß-sheet domain is relatively unstructured. Apparently, these results are related to a fundamental difficulty in studying the conformation of large semi-flexible molecules by a chemical reaction. Clearly, the chemical reaction shows that two reacting groups can touch each other, which does not necessarily mean that their average distance is small.
Only a few physical methods provide direct information on intramolecular distances and fluorescence energy transfer is one of them, suitable for the investigation of macromolecules in solutions. The first attempt in this direction was made two decades ago and was based on the covalent binding of extrinsic fluorophores to protein functional groups. This approach, however, has not been widely used because of possible labeling heterogeneity, structural alteration resulting from the labeling per se and the effect of labeling on the protein folding (Lakowicz, 1983; Cheung, 1991
). Fortunately, towards the end of the 1960s it was found that the reaction of tyrosine (Tyr) with tetranitromethane results in the formation of 3-nitrotyrosine (Lundblad, 1991
). As a consequence, nitrated tyrosine, Tyr(NO2), becomes an acceptor of tryptophan (Trp) electronic excitation energy (Borkman and Phillips, 1985
). This means that the decrease in Trp fluorescence in the presence of Tyr(NO2) depends on the average distance RDA between Trp and Tyr(NO2) residues. The strong dependence of an energy transfer on a donoracceptor distance allows us to measure RDA with high accuracy. If one can nitrate the Tyr residues of the protein chain and measure the decrease in Trp fluorescence following the modification then one can measure the average distance between two definite points of the protein chain. We applied this method to compare the 3D structure of horse apomyoglobin in the native and molten globule states.
The combination of deuterium exchange and 2D-NMR revealed that the native A-, G-, and H-helixes of apomyoglobin become relatively stable at an early stage of protein folding (Jennings and Wright, 1990) and are relatively stable in the equilibrium molten globule (Hughson et al., 1990
). Multi-dimensional NMR spectroscopy provides (Eliezer et al., 1998
) the backbone conformation of residues in the molten globule state, which suggests the localization and probability of these helixes. However, the relative positions of the most stable helixes A, G and H in the molten globule are still questionable. We attempted to obtain this information by measuring the intramolecular energy transfer from Trp residues to Tyr(NO2). Fortunately, horse myoglobin contains Trp7 and Trp14 in the middle of the A-helix, Tyr103 at the beginning of the G-helix and Tyr146 at the end of the H-helix (Figure 1
). The comparison of Trp fluorescence in non-modified and selectively and entirely modified samples permits one to evaluate the average distances from Trp to Tyr residues, i.e. from the middle of the A-helix to the beginning of the G-helix and to the end of the H-helix.
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Materials and methods |
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Horse heart myoglobin was purchased from Sigma. The heme was removed by the method of Teale (1959) followed by gel chromatography with Pharmacia PD-10 columns containing Sephadex G-25. After heme removal, the proteins were dialyzed against water at 4°C and then lyophilized for storage. The contamination of the apoprotein by myoglobin was assessed by spectroscopic means. No significant absorption was observed in the Soret region. The homogeneity of the apomyoglobin preparations was tested by SDSPAGE (Laemmli, 1970). A single band was always observed for the proteins investigated. The protein concentration was estimated by measuring the absorption at 280 nm with a Hewlett-Packard Model 8452A diode-array spectrophotometer. The molar extinction at 280 nm was calculated from the Trp and Tyr content by using extinction coefficients (
) of 5600 and 1250 M1 cm1, respectively.
The reaction to convert selectively Tyr146 into Tyr146(NO2) was similar to that previously described in detail (Rischel and Poulsen, 1995). To nitrate both Tyr residues the protein was dissolved at 6 µM in 0.1 M Tris buffer (pH 7.7) containing 6 M GuHCl. A 150 µl aliquot of a 1 wt% solution of tetranitromethane in ethanol was added to 1700 µl of protein solution. The mixture was left with stirring in the dark for 5 min and thereafter applied to the gel filtration columns equilibrated with water. The proteins were dialyzed against water at 4°C and lyophilized for storage.
The extent of Tyr modification was assessed by spectroscopic means. We used the fact (Riordan et al., 1967) that in an alkane medium 3-nitrotyrosine has an absorption maximum at 428 nm, whereas in an acidic medium the absorption maximum shifts to 360 nm with an isosbestic point at 381 nm (
= 2200 M1 cm1). Figure 2
illustrates the variation of the protein absorption following the modification. The absorption peak of Tyr(NO2) at 360 nm is clearly visible. The calculation of Tyr(NO2) concentration with
381 nm = 2200 M1 cm1 shows that the molar contents of Tyr(NO2) per protein have a value of 1 and 2 for selectively and entirely modified samples, respectively. Further, the efficiency of modification was estimated by mass spectrometry (data are not shown). We found that the mass spectrum of horse apomyoglobin corresponds to the theoretical mass of 16 951. Meanwhile, the mass spectra of selectively and entirely modified samples showed shifts of 45 and 90 units, respectively. The fraction of modified molecules was found to be about 90%. The selectivity of the modification was verified by proteolytic cleavage of the `selectively' modified protein (with only nitrated Tyr146) and mass spectrometry of the resulting fragments essentially as described (Rischel and Poulsen, 1995
). Only the fragments with Tyr146 showed a mass deviation of 45 units in accord with the mass of the NO2 group.
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Steady-state fluorescence energy transfer experiments
Fluorescence spectra were measured with SPEX Fluorolog-2 spectrofluorimeter supplied with DM-3000 software. Excitation was set at 280 nm. Fluorescence was measured in the ratio mode and background fluorescence from a solvent blank was subtracted. Spectra were corrected for the wavelength dependence of the instrument response. Fluorescence quantum yields of the protein in solution were measured by comparison with twice-recrystallized N-acetyltryptophanamide (standard) as follows:
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The efficiency of energy transfer E was calculated as the relative loss of the donor fluorescence due to the interaction with the acceptor (Förster, 1948):
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Results and discussion |
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Direct energy transfer in apomyoglobin
The fluorescence spectra of unmodified apomyoglobin and selectively and entirely modified samples in the native, molten globule and unfolded states are shown in Figure 4. One can see that the Trp fluorescence follows the protein folding in a non-monotonic way. It is substantially stronger in the molten globule state than in the native or unfolded state. This phenomenon is usually observed for apomyoglobins (Irace et al., 1981
; Postnikiva et al., 1991). The increase in Trp fluorescence at the `unfoldedmolten globule' transition reflects Trp residues being hidden from the water environment in the interior of the protein molecule. The further decrease in fluorescence at the `molten globulenative' transition suggests that in the native state Trp residues located in the A-helix may be tightly packed with a quencher.
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We measure the quenching induced by the presence of either only Tyr146(NO2) or both Tyr103(NO2) and Tyr146(NO2). Considering the additive character of the energy transfer, the efficiency per acceptor [Tyr146(NO2) or Tyr103(NO2)] can be calculated in the following way:
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In compact states of apomyoglobin, the energy transfer efficiency was measured to be 60 and 30% for Tyr146(NO2) and Tyr103(NO2), respectively. To obtain the donoracceptor distances one has to assume or directly measure the orientation factor <k2> for the system of interest. For proteins with known 3D structure, the <k2> value might be obtained from data from X-ray and energy transfer experiments. Here we describe one version of this approach. First, for native molecules we measure energy transfer efficiencies E and calculate RDA/R0 ratios by Equation 3. Second, the average distances <RDA> between Trp7 and/or Trp14 and each acceptor are calculated from the crystallographic data reported for horse myoglobin (Evans and Brayer, 1988, 1990
; PDB 1ymb), since it is believed that native apomyoglobin resembles native holomyoglobin (Hughson et al., 1990
). Moreover, it has been established that the difference in the linear dimensions of native apo- and holomyoglobin molecules does not exceed 12% (Kataoka et al., 1995
). We find that the average distances from (Trp7Trp14) to Tyr146 and Tyr103 in native molecules are 22 ± 2 and 24 ± 3 Å, respectively. The scattering shows the difference in donoracceptor distances calculated for each TrpTyr couple. Third, with the RDA/R0 and <RDA> values experimentally obtained we calculate the characteristic radius <R0> for native apomyoglobin and then calculate <k2>eff to be 0.18 (see Equation 4). Another assumption to be made is that the orientation factors have similar values in native and molten globules of apomyoglobin. Fortunately, R0 depends on <k2>1/6, i.e. is a very slow function. Therefore, the uncertainty induced by this assumption should be negligible for <k2> varying from 0.18 (native) to 0.67 (unfolded). Finally, with a <k2>eff value of 0.18 and the energy transfer efficiency measured in the molten globule state we estimate the average distances from the (Trp7Trp14) segment to Tyr146 and Tyr103 to be 22 ± 2 and 27 ± 3 Å, respectively. However, the distance of 27 ± 3 Å between Trp residues and Tyr103 might be overestimated. In fact, the energy transfer efficiency observed in the selectively modified sample is about 60%. This means that the energy transfer in the entirely modified sample may lead to complete quenching of the Trp fluorescence. The fluorescence observed in this sample (Figure 4
) might come from the unmodified fraction of protein molecules.
The most valuable observation in this work is that the direct energy transfer efficiency is almost the same in the molten globule and in the native states of apomyoglobin in selectively and entirely modified samples. The major conclusion to be drawn is that the distances between the middle of the A-helix and the N-terminus of the G-helix and the distance between the middle of the A-helix and the C-terminus of the H-helix in the molten globule state of apomyoglobin are close to those in its native state. This suggests that the molten globule state of horse apomyoglobin has an AGH complex whose overall structure (in terms of `AG' and `AH' separations) is similar to that of the native state. These measurements lend strong support to the hypothesis (Ptitsyn, 1973) of the similarity of the overall architectures of the molten globule and native states. However, to complete this conclusion for apomyoglobins one needs supplementary information about the location of other structural elements. Energy transfer experiments to probe the average distance between G- and H-helixes in the molten globule state of apomyoglobin are in progress.
In summary, this paper demonstrates the feasibility of using steady-state fluorescence data to study the structure of non-native proteins. Owing to the relative simplicity of generating such data, the steady-state energy transfer method may be of widespread usefulness in studies of non-native protein structures in solution, including the structure of folding intermediates.
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Acknowledgments |
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Notes |
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References |
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Received January 7, 1999; revised March 30, 1999; accepted April 16, 1999.