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1 Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain
2 Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200 D, B-3001 Leuven, Belgium
Reprint requests to: Maria Vilanova, Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain; e-mail: dbmvb{at}fc.udg.es; fax: 34-972-41-81-50.
(RECEIVED October 10, 2000; FINAL REVISION January 2, 2001; ACCEPTED January 3, 2001)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.43001.
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Keywords: Ribonuclease A; protein engineering; protein folding; pressure versus heat-induced unfolding; Fourier-transform infrared spectroscopy
Abbreviations: CD, circular dichroism • CFIS, chain folding initiation site • C-terminal, carboxy-terminal • DSC, differential scanning calorimetry • FTIR, Fourier transform infrared • HPLC, high-performance liquid chromatography • MES, 4-morpholine-ethanesulphonic acid • NMR, nuclear magnetic resonance • PCR, polymerase chain reaction • RNase A, bovine pancreatic ribonuclease A • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • UV, ultraviolet
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Introduction |
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Early on, Némethy and Scheraga (1979) proposed several hydrophobic amino acid regions within RNase A as likely CFISs. Residues 106–118 (of 124) are highly conserved among the RNase superfamily (Beintema et al. 1997). This hydrophobic region of the protein, which constitutes a beta-hairpin in the native state (Wlodawer et al. 1988), was predicted to be one of the most probable sites for formation of the initial structure (Matheson and Scheraga 1978). For RNase A, refolding quench flow experiments, combined with two-dimensional (2D) NMR spectroscopy, enabled identification of protons, which are protected early during the refolding reaction (Udgaonkar and Baldwin 1995). The protons of the above-mentioned CFIS are included among these. Recently a hydrogen exchange 2D NMR study of RNase A has identified the slow-exchanging protons and those that exchange by a global unfolding mechanism (Neira et al. 1999). The results of this study, compared with those of Udgaonkar and Baldwin, led the authors to suggest the presence of a putative chain-folding initiation site comprising the four-stranded region, Lys62-Ala64, Cys72-Ser75, Ile106-Cys110, and Val116-His119, together with the face of helix III containing Val54 and Val57.
Using site-directed mutagenesis to engineer RNase A, a set of mainly conservative hydrophobic variants of its 106–118 region was produced. The stability of each variant was compared by pressure- and temperature-induced unfolding and monitored by fourth-derivative UV absorbance spectroscopy (Torrent et al. 1999). The results show the contribution of I106, I107, V108, A109, V116, and V118 hydrophobic residues to overall protein stability, the 108 position being the most important site for stability and the site most destabilized by amino acid replacement. In addition, a comparison of the effect of pressure and temperature on each variant under identical experimental conditions is reported.
Recently, using staphylococcal nuclease as a model protein, pressure effects on the denaturation of variant forms have also been reported by Royer and others (Royer et al. 1993; Vidugiris et al. 1996; Frye et al. 1996; Frye and Royer 1998), showing equally interesting results for correlation studies with temperature denaturation.
Various studies have suggested that pressure and thermally denatured RNase A have compact dimensions and a nonrandom structure, but it still remains uncertain whether there are conformational differences between the two denatured states (Labhardt 1982; Privalov et al. 1989; Sosnick and Trewhella 1992; Seshadri et al. 1994; Tamura and Gekko 1995; Neira et al. 1999; Panick and Winter 2000). In the present study, we combined protein engineering methods and FTIR spectroscopy to characterize further the differences between the pressure- and temperature-unfolded states of RNase A. To this end, the unfolding process of these RNase A variants, I106A/V/L, I107A, V108G/A, V116G, V118G/A, and Y115W, induced by both denaturing agents, was compared with the process presented by wild-type protein. In addition, the impact of amino acid substitutions on the native state of RNase A was also determined. Most of the constructed variants concern ribonucleases bearing nondisruptive deletions, with the sole exceptions of I106L and Y115W amino acid replacements.
It is generally accepted that FTIR spectroscopy is one of the most powerful techniques for determining conformational changes in proteins under equilibrium conditions (Jackson and Mantsch 1995). It has already been used to determine pressure- and temperature-induced changes in the secondary structure of wild-type RNase A (Takeda et al. 1995; Panick and Winter 2000). In the present study, peptide backbone, with its unique signature for ß-sheets and side-chain infrared "marker" bands (Fabian et al. 1993, 1994), were used as conformation-sensitive monitors for following the induced reversible changes in the secondary structure of RNase A wild type and variants. Additionally, marginal changes in protein conformation that may arise from the amino acid substitutions in the hydrophobic 106–118 region were examined. Furthermore, bandfitting of the Fourier-deconvoluted amide I' spectrum (Smeller et al. 1995a) allowed us to follow the variation of the secondary structure elements for the unfolding process of the proteins under study.
The present results show that for RNase A, the two-state assumption is valid for the analysis of either the temperature or the pressure-induced unfolding. They also provide a structural basis for our previous findings concerning the contribution to the stability of RNase A of the hydrophobic interactions in its 106–118 region (Torrent et al. 1999). Further, they provide new insights into the differences between pressure- and temperature-induced changes, and lay the basis for kinetic investigations to probe the role of the region in protein folding.
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A,B). Deconvolution of the original spectrum of the amide I' band showed, within the precision of the method, the presence of component bands at different wavenumbers. The band positions for RNase A wild type after partial hydrogen-deuterium (H-D) exchange are given in Table 1, along with secondary structure assignments based on the work of Byler and Susi (1986). A minor band at 1610 cm-1 is attributable to the amino acid side-chain vibration (Matsuura et al. 1986). It is worth noting that ß-sheets and turns were each assigned to multiple bands. The broad band at about 1651 cm-1 could not be resolved, leaving superimposed the component bands of 
-helices and unordered structures, because of the small wavenumber difference between the amide I' C = O stretch of these two structural components (Dong et al. 1990). Bandfitting of the deconvoluted spectra (Fig. 1B) allowed a semiquantitative estimate of the fractional composition of the secondary structure elements in the native state of wild-type RNase A and variants. The resulting fractional areas (listed in Table 1) of the bands assigned to different types of secondary structure were assumed to represent percentages of these structures in the protein. Percentages of secondary structure content of wild-type protein, from both natural and recombinant sources, are 55% ß-structure, 22% turns and bends, and 22% -helix and unordered. This agrees with the generally accepted secondary structure derived from X-ray crystallographic studies (Wlodawer et al. 1988) and 2D NMR spectroscopic studies (Rico et al. 1989), and supports the assignments made.
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) and V118 sites. Accordingly, the results from curve fitting show, in general, no significant differences in the secondary structure content, under native conditions, of wild-type and variant proteins. Only the small effects of replacements at positions 108 and 118 prove large enough for some secondary structural changes to be discerned with FTIR. The impact on the polypeptide backbone conformation increases in the order, V118G<V108A<V108G. Therefore, we focused on the spectral changes observed in the characterization of the V108G variant, although similar variations apply to the other two variants. As shown in Figure 1C, in the V108G variant there is a small shift of the amide I' band toward a lower wavenumber than that of the wild-type protein. This is especially remarkable for the component band assigned to turn structures, which is shifted from 1686 cm-1 to 1684 cm-1, suggesting a more complete initial hydrogen-deuterium exchange. In particular, the broader amide I' component band at 1637 cm-1 shown for the V108G variant may indicate that fine differences distinguish the hydrogen-bonding pattern of the ß-sheet structures from that of the wild-type protein. Moreover, the increases in intensity at 1651 and 1684 cm-1 bands, assigned to -helix + unordered and turns, respectively, point to a higher percentage of irregular and turn structures in this variant. We noticed differences, although these were not systematically studied, in the rate and extent of H-D exchange between the variants, which suggested differences in the dynamics of the structures.
Pressure and temperature dependence of the amide I' FTIR spectrum
Pressure- and temperature-induced changes in the deconvoluted amide I' region of the IR spectrum were followed to obtain information about protein structure changes. Typical alterations in the amide I' region of the V118G variant on increases in pressure or temperature are shown in Figure 2, panels A and B, respectively. For all the proteins analyzed, cooperative spectral changes are observed when pressure or temperature is increased, although gradual absorbance changes occurred before the unfolding transition. At low pressures or temperatures, a general shift of the amide I' band around 1637 cm-1 toward a lower wavenumber is observed. As no changes in intensity of this main compositional band are observed under these conditions, these alterations may be assigned to enhanced H-D exchange in the interior of the protein. In contrast, the V108G variant showed only a very small shift, suggesting a more complete initial H-D exchange. As pressure or temperature is increased for all the variants and wild-type protein, a co-operative decrease in band intensities is observed, indicating the disappearance of native structures. There is also an increase in band intensity around 1645 cm-1, indicating an increase in the amount of unordered structure. At high pressure or temperature, it seems that the unordered structure dominates in the final stage of protein unfolding, although the proteins nevertheless retain some secondary structure. This is consistent with previous results on the wild-type protein monitored using CD (Labhardt 1982; Yang et al. 1986; Seshadri et al. 1994), dynamic light scattering (Nicoli and Benedek 1976), small-angle X-ray scattering (Sosnick and Trewhella 1992), FTIR (Anderle and Mendelsohn 1987; Sosnick and Trewhella 1992; Panick and Winter 2000), compressibility (Tamura and Gekko 1995), and NMR H-D exchange data (Talluri and Scheraga 1990; Neira et al. 1999). Interestingly, comparison between IR spectra of the pressure-denatured proteins and of the corresponding thermally denatured proteins (Fig. 3A,B) shows similar amide I' band profiles, but with the spectral maxima centered at different wavenumbers of the amide I' vibrational mode.
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According to the changes observed in the amide Iv band, secondary structure elements are disrupted cooperatively, and there is one main transition, preceded and followed by (near linear) curves, when the pressure or temperature is increased. This is also observed in the small shifts that take place in the Tyr side bands (Fig. 4).
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All unfolding transition curves were fitted to a two-state thermodynamic model combined with sloping functions for the native and denatured states as described previously (Pace 1990). The results show that all equilibrium-unfolding profiles are shifted to lower pressures or temperatures than in the wild-type protein. The amino acid substitutions decreased the stability of the protein by different amounts. The midpoint transition values, P1/2 and T1/2, obtained from the induced changes in the amide I' region on increasing pressure and temperature, are listed in Table 2. They give a consistent idea of the relative stabilities among the different sites and among different variants at the same site. The figures range from 151 MPa for V108G to 743 MPa for wild-type RNase A, and from 37°C for V108G to 62°C for wild-type protein, respectively.
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). They correlate well with those recently found under comparable experimental conditions (at 1 mg/mL) using fourth-derivative UV absorbance spectroscopy (Torrent et al. 1999). Both Tyr band and UV spectroscopy were used as local monitors that detect changes in the tertiary structure along the denaturation process. These data indicate a simultaneous breakdown of secondary structure elements and pronounced changes in the environment of Tyr side-chain groups, suggesting a highly cooperative unfolding process. However, as is to be expected, the T1/2 values obtained in D2O buffer were higher than those derived from the UV data in H2O buffer. It is well known that proteins in D2O solutions show higher T1/2 values than those found in H2O solutions (Talluri and Sheraga 1990; Yamamoto and Tasumi 1991; Fabian et al. 1994). The data given in Table 2, consistent with our previous results, also suggest that among the variants at each site, stability decreases in the order Ile>Val>Leu>Ala>Gly, and that two particular residues, I106 and V108, are the ones most destabilized by amino acid replacement. Amino acid replacements that produce the greatest protein destabilization (i.e., V108G) seem to affect the residual secondary structure of pressure and temperature unfolded states in a different way.
The spectra of the pressure and temperature denaturated states of RNase A wild type and V108G variant are compared in Figure 3, panels A and B, respectively. From the temperature-denaturated spectral outlines, the amino acid change seems to lead to a more Gaussian amide I' band and to the disappearance of the shoulder at around 1670 cm-1. In contrast, the pressure-denaturated spectral outlines for the two proteins are practically identical.
To determine the trends in the conformational changes of the protein backbone for RNase A wild type and its variants in terms of variation of secondary structure elements on increasing pressure and temperature, the deconvoluted spectral progressions with both agents were curve fitted (Smeller et al. 1995a). FTIR spectroscopy is particularly sensitive to changes in ß-strands of proteins (Fabian et al. 1994). As listed in Table 1, three component bands (at 1624, 1637, and 1678 cm-1) account for this secondary structural element. Among them, the intensity of the 1637 cm-1 band is the strongest. In fact, about half of the whole fitted area of the deconvoluted spectrum of native RNase A belongs to this ß-sheet band. Because the amino acid substitutions engineered in this work were performed in a region of ß-sheet structure of RNase A, most significant changes in the recorded IR spectra were expected to arise from this secondary structural element. Therefore, we focused on the percentage variation of the main ß-sheet component of the amide I' band at 1637 cm-1 and the broad band at 1651 cm-1 that indicates the appearance of unordered structures. Both bands show abrupt and simultaneous changes of intensity on increases in pressure and temperature. On analysis of the whole set of variants, the stability results obtained basically confirm those obtained from the shape of the amide I' band.
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Native and denatured states
Judging from the amide I' band contour at 12°C and 0.1 MPa, all the variants have a similar folded conformation to the wild-type protein, with the exception of variants V108G, V108A, and V118G, in which subtle conformational differences seem to be present. This observation is consistent with data from CD measurements, although significant structural differences were only found in the V108G variant (Torrent et al. 1999).
The pressure- and temperature-denatured states of all the ribonucleases characterized in this study show some secondary structure. However, it is noteworthy that their spectral maxima are centered at different wavenumbers of the amide I' vibrational mode. Whereas the spectrum at 1200 MPa is centered at 1640 cm-1, the spectrum at 85°C is centered at 1650 cm-1 (Fig. 3). A previous study involving wild-type RNase A offers some insight into the significance of this spectral shift (Takeda et al. 1995). The authors suggest a difference in hydration of the backbone peptide groups between the pressure- and temperature-unfolded states. Apparently, pressure or temperature leads to different unfolded states of the protein. The higher intensity at 1630 cm-1 in the pressure-unfolded state suggests a higher ß-sheet content. The higher intensity at 1670 cm-1 in the temperature-unfolded state suggests a higher content of turns. This seems to corroborate numerous studies in which the pressure-induced protein unfolding appears to lead to a molten globule state (Zhang et al. 1995; Yamaguchi et al. 1995) in which native-like secondary structure is retained but solvent is admitted to the core regions of the protein. In contrast, the thermally unfolded state appears to be more extensively unfolded (Zhang et al. 1995).
Stability
The pressure and temperature stabilities of the variants are indicated by comparison of the P1/2 and T1/2 values (Table 2). Unfolding midpoint values obtained from monitoring unfolding transitions using both peptide backbone and Tyr side-chain infrared "marker" bands (Arrondo et al. 1988; Fabian et al. 1993, 1994) are consistent with each other and are also consistent with the hypothesis that the pressure- and temperature-induced unfolding of RNase A, under equilibrium conditions, is an overall two-state and highly cooperative process, involving all structural components between folded and unfolded species. This is in line with data obtained by fourth-derivative UV absorption spectroscopy (Torrent et al. 1999) and by DSC analysis (Coll et al. 1999). The Tyr band shift observed in the IR and the UV absorbance change seems to reflect the same molecular events during protein unfolding. Monitoring of RNase A wild-type unfolding gave similar results (Reinstädler et al. 1996). The fact that the spectral changes occur at about the same pressure and temperature using low and high protein concentration regime techniques, UV and FTIR respectively, is a strong indication that the observed unfolding events are intramolecular.
It is notable that all variants have lower transition midpoint values than wild-type protein. Of particular interest is the dramatic pressure and temperature destabilization of the V108G variant. Its pressure- and temperature-unfolding midpoint values decrease by 592 MPa and 25°C, respectively, compared to wild-type protein. These results suggest that V108 is an important residue for the structural integrity of the RNase A fold. The exchange of the amide and side-chain hydrogens with solvent deuterons observed for native V108G, possibly higher than for wild-type protein, could be associated with greater conformational flexibility and a more open or dynamic structure. Both observations (lower pressure and temperature stability and an increased protein flexibility) suggest that this amino acid substitution could potentially leave a cavity in the variant protein, which several investigators (Buckle et al. 1996; Fusi et al. 1997), using similar point amino acid substitutions in other proteins, have addressed.
Recently, we followed the thermal denaturation of wild-type RNase A and its variants by DSC (Coll et al. 1999) and found a ratio between calorimetric and van't Hoff enthalpies (R value) near 1 for wild-type RNase A and most of the variants. The sole exception was the variant at position 108, which showed R values lower than unity. This has been interpreted as a proof of existence of self-association between molecules during denaturation, specifically in the case of V108G RNase A variant. This effect is reversible on cooling. Surprisingly, when the same variants at position 108 were analyzed, both by pressure- and temperature-induced unfolding, no aggregation process was found. Although this may be because of the different techniques used to monitor the unfolding process, it stresses the particular dynamic nature attributed to the V108G RNase A variant.
In our previous study (Torrent et al. 1999), we showed that part of the observed destabilization of the different variants is accounted for largely by the change in the nature of the side chain (i.e., the change in core residue volume and hydrophobicity). The remaining effect on stability was found to result from the environment surrounding the different residues, primarily the role of van der Waals interactions, which are generally accepted to have a strong dependence on the packing density and the solvent exposure in the native structure. These results are consistent with the greater effects found at the Val108 and Ile106 sites, which are the most destabilized by amino acid substitution and thus the two most important residues for stability. Their importance is emphasized by their high conservation in the RNase sequences (Beintema et al. 1997).
As determined by 2D NMR spectroscopic studies of native wild-type RNase A, the backbone amide protons buried or involved in hydrogen-bonded structures, such as ß-sheets and -helices, are protected strongly against exchanges with solvent deuterons (Zhang et al. 1995; Talluri and Scheraga 1990; Robertson and Baldwin 1991; Nash et al. 1996; Neira et al. 1999). Exchange-induced band shift has been shown at pressure or temperature below the cooperative unfolding of secondary structure for almost all variants. The major amide I' band at 1637 cm-1 shifts toward lower wavenumbers when a pressure or temperature increase leads to a complete H-D exchange. This band is skewed pronouncedly in all variants, except for V108G, in which a marked difference in the rate of exchange seems to be present, probably because of slightly higher flexibility. Backbone amide protons associated with turns and random coil structures appear to exchange more under conditions that favor the native state, as small shifts of the corresponding amide I' component bands are observed. The results are consistent with recent H-D exchange experiments in RNase A, which have posited that some amide protons located within a putative folding initiation site, primarily comprising a four-stranded ß-sheet region, may account for the slow exchange in the unfolded protein observed (Neira et al. 1999). Similarly, Talluri and Scheraga (1990) reported that temperature-denatured RNase A has a residual structure in the vicinity of the C-terminal CFIS, which indicates the presence of short- and long-range interactions. Thus, because it appears that the pressure and thermally denatured states of RNase A have substantial secondary structure, probably primarily in the CFIS region, it is reasonable to assume that amino acid substitution in this region will lead to a further loss of the remaining structure, as mentioned above. The results fit well the hypothesis that the persisting protons participate constitutively in protein structure stabilization (Backmann et al. 1996), and also fit the conclusion that the hydrophobic residues of the postulated CFIS appear to promote secondary structural contacts, which are important during folding of the ß-sheet region. It seems likely that this region consolidates around the weak secondary structure of the ß-hairpin under study. The importance of these hydrophobic residues to the stability and structure of the whole protein reinforces the idea that the ß-hairpin in RNAse A is an important structural element for attaining a native-like fold.
Comparison of pressure and temperature unfolding
Measurements of effects of amino acid substitutions on RNase A stability have been studied to date by thermal and chemically induced unfolding. We compare here how the changes in amino acid hydrophobicity and volume have affected the pressure- and temperature-induced denaturation of the different RNase A variants. It is well known that pressure affects the volume of the system, leads to changes in protein and solvent organization, and drives protein equilibrium toward the lower volume unfolded state. However, temperature increase affects both thermal energy and the volume of protein and solvent. The results reveal a surprising similarity between both processes of denaturation (Fig. 2).
In line with our previous equilibrium studies, the relative stability of the various RNase A variants is virtually the same for pressure- and heat-induced unfolding. That is, a plot of P1/2 against T1/2 can be fitted by a straight line (Fig. 5). The linear correlation between T1/2 and P1/2 follows from the assumption that the unfolding is a two-state process. In this case the change in enthalpy is a linear function of T1/2 (Elwell and Schellman 1977). For ribonuclease this relationship holds for a large number of experimental approaches (Pace et al. 1999):
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At the molecular level the correlation between P1/2 and T1/2 found in the pressure and temperature experiments may be explained by the particular parameter we are varying, that is, the hydrophobicity of the CFIS. In other words, this suggests that pressure and heat have comparable effects on the hydration of hydrophobic residues. In addition, the multiplicity of weak noncovalent interactions often leads to nonequivocal conclusions as to the predominant role of one type of interaction (Cooper 1999).
A comparative analysis of the amide I' region of the infrared spectrum of pressure- and temperature-denatured RNase A wild type and variants indicates that they are very similar. Indeed, whereas heat denaturation leads in most proteins to the formation of irreversible intermolecular ß-sheet structure and molecular aggregation, our data clearly indicate the absence of aggregation in all the thermally denatured RNase A variants. This is shown by the absence of an increase in intensity of the amide I' band around 1615–1620 cm-1. It has been reported previously that RNase A does not aggregate on unfolding at high temperature (Clark et al. 1981). Consequently, both pressure and thermally denatured RNase A lead to a reversible protein state that contains a predominance of unordered structure and some residual structure.
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Mutagenesis, protein expression, and purification
The gene that encodes RNase A (DelCardayré et al. 1995) was used as a template for mutagenesis by PCR following a method described elsewhere (Juncosa-Ginestà et al. 1994). The mutated and wild-type RNase A genes were transferred into an expression vector, expressed, and purified as described previously (Torrent et al. 1999). Protein purity and homogeneity were confirmed by SDS-PAGE and by cation exchange as well as by reversed-phase HPLC, using a Mono S HR 5/5 and a 214TP Vydac C4 column, respectively.
Preparation of samples and FTIR studies
Lyophilised pure proteins were dissolved to a concentration of 75 mg/mL in 50 mM MES buffer at pD 5.0 for pressure experiments, and in 50 mM sodium acetate buffer pD 5.0 for temperature experiments. pD was measured by adding 0.4 units to the pH value (Glasoe and Long 1960). These buffers were selected for their relatively small pressure and thermal pD dependencies, respectively (Kitamura and Itoh 1987). Samples were dissolved and stored overnight at room temperature to ensure that the H-D exchange reached a static regime. IR spectra were recorded as a function of temperature at atmospheric pressure, or as a function of pressure at 20°C. Spectra were recorded using a Bruker IFS66 FT-IR spectrometer (Karlsruhe) equipped with a liquid nitrogen-cooled mercury-cadmium-telluride detector. Infrared light was focused on the sample by a NaCl lens. The sample compartment was purged continuously with dry air. 250 interferograms were coadded at a resolution of 2 cm-1. For pressure measurements, the protein solution was mounted in a stainless steel gasket of a diamond anvil cell obtained from Diacell Products. The initial gasket thickness was 0.05 mm and the hole diameter 0.65 mm. Pressure was measured with BaSO4 by following the shift of the 983 cm-1 band (Wong and Moffat 1989). Pressure was increased at a rate of about 100 MPa per hour. For the temperature experiments, protein solutions were placed between a pair of CaF2 windows separated by a gasket of 0.03 mm. The cell was controlled by a Graseby/Specac low-voltage heating system. Temperature was increased at a rate of 0.2°C/min. Secondary structure prediction was based on a combination of Fourier self-deconvolution with band fitting (Byler and Susi 1986; Harris and Chapman 1995). Fourier self-deconvolution, a mathematical technique of bandnarrowing, was performed with the Bruker software. A half bandwidth of 21 cm-1 and an enhancement factor (K) of 1.7 were used (Smeller et al. 1995b).
To compare qualitatively the spectra of different variants, we normalized each spectra by use of the corresponding minimum and resulting maximum absorbance. First, each native or denatured spectrum was subtracted from its minimum absorbance. Second, each resulting spectrum was then divided by its maximum absorbance, yielding a normalized spectrum from 0 to 1.
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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