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1 Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA
2 National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 157 84 Athens, Greece
Reprint requests to: George J. Thomas, Jr., Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA; e-mail: thomasgj{at}umkc.edu; fax: (816) 235-1503.
(RECEIVED September 27, 2002; FINAL REVISION December 13, 2002; ACCEPTED January 7, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0234103.
3 This article is part LXXXI in the series Raman Spectral Studies of Nucleic Acids. 
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-helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain ß-stranded structure in excess (eight ± two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm-1), Ala (908 cm-1), Arg (1082 cm-1), and Pro (1457 cm-1). The Ala marker undergoes a substantial shift (908 
893 cm-1) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the -helical core exhibits robust resistance to exchange. A quantitative study of NH ND exchange exploiting newly identified amide II' markers of helical (1440 cm-1) and nonhelical (1472 cm-1) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins. Keywords: Architectural protein; structure; conformation; DNA binding; DNA recognition; Raman spectroscopy; amide II'; hydrogen exchange
Abbreviations: CD, circular dichroism • CM, carboxymethyl • ds, double-stranded • EDTA, ethylenediamine tetraacetic acid • GuDCl, guanidinium deuteriochloride • GuHCl, guanidinium hydrochloride • H/D, hydrogen/deuterium • HU, histone-like prokaryotic DNA-binding protein • HUBst, protein HU from Bacillus stearothermophilus • IHF, integration host factor • UVRR, ultraviolet-resonance Raman • LB, Luria-Bertani • IPTG, isopropyl-ß-D-thiogalactoside; PLA, poly-L-arginine • PMSF, phenylmethylsulfonyl fluoride • SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis • Tris, tris(hydroxymethylamino)methane
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Introduction |
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HU proteins typically contain ~90 residues (molecular weight, ~10 kD). Both homodimeric and heterodimeric forms have been described (Drlica and Rouviere-Yaniv 1987). The structure of homodimeric HU from Bacillus stearothermophilus (HUBst) has been determined in both solution and crystal states (Boelens et al. 1996; White et al. 1999). The subunit N-terminal domain consists of two -helices, which are interleaved with corresponding -helices of a partner subunit to form a four-helix platform. The C-terminal domain of each subunit forms a largely antiparallel ß-stranded fold, which projects from the helix platform. The two C-terminal folds of the dimer do not interact with one another but diverge to form arms (ß-arms) suitable for grasping the DNA double helix (Fig. 1
; Boelens et al. 1996; White et al. 1999).
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15N-NMR chemical shifts of the HUBst:DNA complex indicate that several other side-chains on the inner surface of the ß-arms (Met, Ile, Val, Asn, Gln) are also involved in DNA interactions (Boelens et al. 1996). Although no high-resolution structure is available for any HU:DNA complex, Pro, Met, and Ile side-chains are found consistently at the tips of the ß-arms in the DNA-free structures. It has been proposed that these residues may function to "pry open" the minor groove and bend the DNA toward the major groove (Rice 1997; White et al. 1999). This proposal is supported by sequence and structure similarities between HUBst and the histone-like integration host factor (IHF). IHF is presently the only histone-like protein for which an X-ray crystal structure has been determined in a complex with DNA (Rice et al. 1996). IHF binds DNA both specifically and nonspecifically (Rice 1997; Holbrook et al. 2001), whereas most other histone-like proteins, including HUBst, bind nonspecifically (Drlica and Rouviere-Yaniv 1987). Differences in DNA-binding specificity among various histone-like proteins may be caused by differences in side-chains and their orientations along the putative DNA interface. Additional structural studies are required to identify such differences.
In this study, we report and interpret Raman and ultraviolet-resonance Raman (UVRR) spectra of HUBst. The data provide definitive vibrational assignments for the HUBst solution structure and represent the first comprehensive Raman analysis of a protein that is notably deficient in aromatic amino acid residues. The absence from HUBst Raman and UVRR spectra of the otherwise intense spectral markers expected from Tyr and Trp side-chains facilitates the identification of novel Raman markers of main-chain secondary structure and of key nonaromatic residues, including Ala, Arg, and Pro, which are of significance in DNA recognition. Finally, the present study establishes a foundation for future exploitation of Raman and UVRR spectroscopy to probe the structure and dynamics of HUBst: DNA complexes (D. Serban and G.J. Thomas, Jr., in prep.) and to further characterize DNA structural perturbations induced by specific and nonspecific DNA-binding proteins (Benevides et al. 2000a, b).
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, trace A) is distinguished by a strong and broad amide I band centered at 1658 cm-1 and a complex amide III profile, which includes a major peak at 1250 cm-1 and weaker shoulders at ~1241, 1279, and 1308 cm-1. These are the Raman amide I and amide III features expected for a protein fold consisting of -helix, ß-strand, and irregular conformations (Miura and Thomas, Jr., 1995; Tuma and Thomas, Jr., 2001). A quantitative estimate of the HUBst secondary structure based on the Raman amide I reference intensity profile method (Berjot et al. 1987) indicates 36 ± 2% -helix, 25 ± 3% ß-strand, and 38 ± 3% irregular conformations (loops, turns, coils, and other aperiodic structures) for the dimer at 20°C. This method makes use of the fact that secondary structure contributions to the amide I band profile from -helical, ß-stranded, and irregular conformations, respectively, occur primarily near 1650 to 1655 cm-1, 1665 to 1680 cm-1, and 1655 to 1665 cm-1 (Miura and Thomas, Jr., 1995). The amide III envelope, which likewise comprises contributions from -helix (1279 and 1308 cm-1), ß-strand (1241 cm-1), and irregular conformations (1250 cm-1), is consistent with the amide I analysis. The Raman results are also in accord with the -helix content of HUBst reported in previous NMR and X-ray studies (Boelens et al. 1996; White et al. 1999; Raves et al. 2001) and with the CD profile of HUBst in dilute solution (data not shown).
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ND exchange ND exchange in proteins provides a basis for identifying domains that participate in inter-subunit contacts or undergo conformational changes along folding and assembly pathways (Tuma and Thomas, Jr., 1997; Tuma et al. 1999). Here, we use the method to distinguish HUBst domains that are either exchanged or protected in the native state. Figure 2
compares Raman spectra of the natively folded HUBst dimer in three isotopomeric forms: Trace A is the Raman signature of fully protiated (nonexchanged) HUBst; trace B is that of partially deuterated (natively exchanged) HUBst; and trace C is the signature of HUBst that is fully deuterated (refolded after complete exchange in 6 M GuDCl). Trace D (B - A) shows the computed Raman difference spectrum representing native exchange. The additional Raman differences resulting from exchange of domains that are protected in the native state are revealed in trace E (C - B). The cumulative effects of both native and unfolded exchanges are shown in trace F (C - A). All of the assigned Raman amide I/I' and amide III/III' markers (Table 1) are perturbed as expected by the peptide NH ND exchanges of Figure 2. The salient findings and their interpretations are as follows.
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1654 cm-1) is resolved in the difference spectrum of Figure 2D as a peak/trough feature at 1645/1674 cm-1. The trough component demonstrates that native-state exchange occurs predominantly at sites that are located in regions of ß-strand or irregular secondary structure, that is, in non–-helical domains of the native HUBst fold. On the other hand, the peptide sites that are protected in the native state but unprotected in the unfolded state occur primarily in -helical domains, by virtue of the reduced amide I band intensity (difference trough) revealed at 1653 cm-1 in the difference spectrum of Figure 2E. The relative areas of the amide I difference troughs in Figure 2, D and E (~4:1), show further that in native HUBst the non–-helical structure significantly exceeds the -helical structure.
Secondary structures of natively exchanged and protected sites of HUBst can also be deduced from amide III/III' difference features. Native exchange (Fig. 2D) is manifested in intense trough/peak features near 1240/986 cm-1 (ß-strand) and 1255/924 cm-1 (irregular) and a weaker feature near 1305/951 cm-1 (-helix). Conversely, subsequent exchange of unfolded HUBst (Fig. 2E) is dominated by the trough/peak pattern at 1300/946 cm-1 (-helix). The amide III/III' data are thus in agreement with the amide I/I' results in demonstrating that exchange of the native HUBst fold occurs largely in non–-helical regions, whereas the exchange-protected sites are located predominantly in -helical regions (Miura and Thomas, Jr., 1995).
The relative intensities of amide III' difference peaks in the 900- to 1000-cm-1 interval may also be used to estimate the populations of -helical and non–-helical sites undergoing peptide exchange (Tuma et al. 1996, 2001; Tuma and Thomas, Jr., 1997). We find that the integrated intensity of the amide III' difference envelope in Figure 2D exceeds that of Figure 2E by roughly 4:1, implying in agreement with the amide I results that ~80% and ~20%, respectively, of HUBst residues are exchanged and protected in the native structure.
Many other difference features are also observed in the computed difference spectra of Figure 2. The most prominent of these is the Raman amide II' band, which (unlike the vanishingly weak amide II band) exhibits significant intensity in Raman spectra of many deuterated proteins (Bandekar 1992; Tuma et al. 1996; Tuma and Thomas, Jr., 1997; Lee and Krimm 1998). In natively exchanged HUBst, the intense difference peaks near 1440 and 1473 cm-1 (Fig. 2D) are assigned collectively to amide II' modes of all secondary structure types. The residues protected in the native structure but exchanged in the unfolded structure generate amide II' peaks at 1427 and 1458 cm-1. Although reliable secondary structure assignments have not previously been proposed for these amide II' markers, we propose on the basis of the amide I/I' and amide III/III' results that the -helical (exchange-protected) sites of HUBst generate amide II' intensity in the 1427- to 1440-cm-1 interval, whereas non–-helical (unprotected) sites generate amide II' intensity in the 1458- to 1473-cm-1 interval.
Raman markers of alanine and effects of peptide exchange
Of additional interest in Figure 2 are difference bands resulting from the exchange of peptide sites of specific amino acid side-chains. Two types of exchange-sensitive bands of the side-chains can be distinguished: (1) bands representing localized vibrations of exchangeable sites (e.g., arginyl guanidinium modes that shift with N
H or N
H deuteration, discussed below), and (2) bands representing coupled side-chain/main-chain vibrations. A well-studied example of the latter is the alanyl marker near 908 ± 2 cm-1, which involves both C-Cß stretching and peptide N-H bending and therefore exhibits a small, but significant, shift to lower wavenumber (893 ± 2 cm-1) with alanyl peptide deuteration (Sutton and Koenig 1970). The 908 893 cm-1 shift represents a specific Raman signature of alanyl NH ND exchange. Thus, the trough/peak at 908/983 cm-1 in Figure 2F provides a benchmark for comparison of protected (Fig. 2E) and unprotected (Fig. 2D) alanyl NH sites. The data of Figure 2 show that the majority of alanyl NH sites are exchange-protected in the native structure. This is consistent with exchange characteristics of the Raman amide bands and with the crystal structure of HUBst, which shows that alanines are located predominantly in regions of -helical secondary structure.
Raman markers of arginine
Four of the five arginines of HUBst (Arg 53, Arg 55, Arg 58, Arg 61) are located in the DNA-binding domain. A fifth arginine (Arg 37) is located in helix 2 (Fig. 1). To identify Raman markers of these side-chains and signatures of their guanidinium exchanges (NH ND, NH ND), we examined Raman spectra of L-arginine and poly-L-arginine (PLA) in H2O and D2O solutions. The spectrum of L-arginine in H2O exhibits a pair of arginyl markers at 1054 and 1086 cm-1 (Fig. 3, top panel). In PLA these occur at 1072 and 1091 cm-1, respectively (Fig. 3, bottom panel). The bands may be assigned primarily to coupled C-C and C-N skeletal stretching vibrations of the arginyl side-chain, although the effect of peptide bond formation indicates modest coupling with the peptide group in PLA and/or with amino and carboxyl groups in L-arginine. Nevertheless, significant involvement of the guanidinium group is indicated by the fact that the arginyl markers suffer large intensity changes in D2O solutions of L-arginine and PLA. The deuterated analogs both exhibit an intense band at 907 ± 1 cm-1 and weaker satellites of higher and lower wavenumber values. In the case of PLA, the trio of deuterated arginyl markers (831/907/943 cm-1) is clearly evident in the difference spectrum of the lower panel of Figure 4. Because the 907 cm-1 Raman marker is identical in L-arginine and PLA, the band is unlikely to involve significant coupling with the peptide group and may be regarded as diagnostic of the CßH2–C
H2–C
H2–ND–C
(ND2)2 moiety.
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), which is at the center of gravity of the expected arginyl markers. The band center and deuteration behavior both support assignment to the five arginines per subunit. The apparent coalescence of the expected PLA doublet (1072/1091 cm-1) into a single band in HUBst (1082 cm-1) indicates sensitivity to the side-chain orientations and local environments. Although guanidinium group deuterations in native HUBst are expected to shift the 1082 cm-1 band to 907 cm-1, with the attendant appearance in Figure 2D of a difference peak at 907 cm-1, no peak is detected. This can be attributed to cancellation of the expected positive difference intensity by the negative difference intensity accompanying deuteration of the natively exchanged alanines, as discussed in the last paragraph of the preceding section.
Raman markers of other HUBst side-chains
In addition to alanine and arginine, the four phenylalanines per subunit are prolific contributors to the Raman spectra of HUBst (Table 1). The only other amino acid type for which a specific spectral contribution can be proposed is methionine. Thus, the weak band at 701 cm-1 and the medium intensity band at 743 cm-1 are likely to involve C-S stretching vibrations of the Met 1 and Met 69 side-chains. Previously proposed correlations (Nogami et al. 1975a,b) indicate that the torsion 
3 (Cß–C–S–C) of each Met side-chain is in the trans range. The 701-cm-1 marker is well isolated from other bands and might provide a useful probe of changes in methionine local environment with DNA binding. Met 69 has been proposed as a DNA recognition element, possibly intercalating into the minor groove of bound DNA (White et al. 1999).
Although aliphatic side-chains make many additional contributions to the HUBst Raman spectra of Figure 2, their bands overlap one another extensively, and detailed amino acid assignments are not feasible. Several such bands and their group vibrational characteristics are listed in Table 1.
UVRR markers of HUBst secondary structure and side-chains
The 229-nm excited UVRR spectra of HUBst in H2O and D2O solutions are compared in the top panel of Figure 4. The dominant effect of native-state deuteration, as reflected in the UVRR spectrum, is enhancement of the amide II' intensity. This is represented by the twin difference peaks at 1442 and 1472 cm-1, which result from exchange of irregular and ß-strand conformations, respectively. In accord with previous studies of model compounds (Austin et al. 1993; Chi et al. 1998), the diminished amide I intensity at 1674 cm-1 and diminished amide II intensity at 1561 cm-1 are also attributable to native exchange of the non–-helical peptide sites. Exchange-protected -helical sites are identified by the UVRR amide II -helix marker that persists at 1553 cm-1 in the D2O solution spectrum of Figure 4. Other peaks and troughs in the Figure 4 difference spectrum are of marginal amplitudes and may reflect experimental error or slightly different UVRR cross sections for side-chain markers in the nondeuterated and deuterated forms of HUBst.
Intensities of the UVRR amide II bands in native and natively exchanged HUBst (Fig. 4, top panel) allow a semiquantitative estimate of the extent of peptide NH ND exchange. Thus, the amide II marker (1561 cm-1) of HUBst in H2O solution, which comprises contributions from all types of secondary structure, is replaced in the natively exchanged protein by an intense amide II' doublet (difference peaks at 1442 and 1472 cm-1; Austin et al. 1993). Peptide sites that remain protected from native exchange account for the residual amide II intensity at 1553 cm-1 (Chi et al. 1998). These results indicate that ~80% of the peptide sites undergo NH ND exchange, in agreement with the off-resonance Raman data of Figure 2. As noted in the next section, the prominent UVRR band observed at 1457 cm-1 for HUBst in H2O solution (Fig. 4, top panel, middle trace) is owing to proline residues.
UVRR marker of proline
The UVRR spectrum of HUBst in H2O reveals an intense and broad band near 1457 cm-1 (Fig. 4, top panel, middle trace). On the basis of 240-nm UVRR studies of prolyl-containing peptides, Takeuchi and Harada (1990) assigned such a band to the imide II mode of the X-Pro peptide linkage. To confirm this assignment for 229-nm UVRR excitation, we obtained spectra of the prolyl-rich peptide bradykinin (Fig. 4, lower panel). The data of Figure 4 indicate that the 1457-cm-1 band of HUBst is the counterpart of the imide II mode of bradykinin (1465 cm-1). The lower wavenumber of the imide II marker in HUBst can be attributed to weaker hydrogen bonding of prolyl C=O sites in HUBst vis-à-vis bradykinin. (Limits observed for the imide II mode are ~1445 cm-1 for a non–hydrogen-bonded C=O group and 1485 cm-1 for a very strongly hydrogen-bonded C=O acceptor; Takeuchi and Harada 1990). Accordingly, the imide II marker of HUBst at 1457 cm-1 is diagnostic of relatively weak prolyl C=O interactions in the native protein.
The bradykinin UVRR data of Figure 4 show that with 229-nm excitation, the prolyl imide II marker is intrinsically more intense than phenylalanyl markers near 1604 and 1003 cm-1. The absence of any deuteration shift indicates further that the marker represents a highly localized vibration of the proline moiety. We note that the UVRR band intensity ratios for Pro and Phe markers in the H2O solution spectrum of HUBst are as expected for the Pro:Phe residue distribution (1:1) in the protein. In addition, all of the UVRR intensity increase observed in the 1440- to 1480-cm-1 interval accompanying HUBst deuteration can be attributed with confidence to nonprolyl peptide exchange (i.e., amide II').
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-helical core (Fig. 1; Boelens et al. 1996). The NMR structural model contains 37% -helix and ~16% ß-strand. The remaining 47% of the NMR structure is presumed to consist of nonrepetitive or irregular conformations. The -helix content in the X-ray crystal structure is in agreement with the NMR solution structure (White et al. 1999), although the X-ray structure is not of sufficient resolution to resolve details of ß-strand or other conformations in the C-terminal arms. Quantitative analyses of the Raman amide I (Berjot et al. 1987) and amide III' (Tuma and Thomas, Jr., 1997) data of Figures 2 and 3 confirm the high -helix content of the solution structure and indicate considerably more ß-strand than inferred by NMR (26% versus 16%). The difference between the NMR- and Raman-based models represents eight (± two) additional residues per subunit in regions of ß-strand at the expense of irregular conformations. Because the nonhelical residues are located principally in the C-terminal arms of the subunit, we propose that these arms are more highly ß-stranded than inferred initially by NMR. A recent refinement of both the NMR (Boelens et al. 1996) and X-ray (White et al. 1999) models supports this hypothesis and attributes the initial under-assessment of ß-strand content to the overall mobility of the C-terminal arms (Raves et al. 2001).
Dynamics and interactions of the HUBst dimer
Measurements of peptide H/D exchange provide a window into protein intramolecular dynamics and the surface area impacted by quaternary interactions (Englander et al. 1996; Englander 2000). We have found that ~80% of the peptide sites of the HUBst dimer undergo NH ND exchange. This is surprising when compared with the percentage (17%) of dimer surface area that is expected to be exposed to solvent. The ratio of exposed to total subunit area is estimated as 0.167, using the relation of Miller et al. (1987), that is, 4.5 • M0.76/1.48 • M = 4840 Å2/28,860 Å2, where M is the subunit molecular weight of 9.75 kD (Wilson et al. 1990). The much greater than expected exchange limit of the HUBst dimer, which reflects substantial exchange of -helical regions as well as complete exchange of nonhelical regions, implies that peptide exchange dynamics extend to -helices at the subunit interface (Englander et al. 1996; Englander 2000). Exchange at the interface presumably reflects pleomorphism in the native dimer that may be required for efficient non-specific binding to DNA. Similar NH ND exchange characteristics have been observed for other protein quaternary assemblies (Tuma et al. 1996, 1998, 1999).
Raman markers of residues conserved among HU proteins
The Raman signature of HUBst exhibits identifiable spectral markers (Table 1) of residues that are conserved among many bacterial histone-like proteins, including arginines (Arg 53, Arg 55, Arg 58, Arg 61) that line the DNA interface and prolines (Pro 63, Pro 72) and methionine (Met 69) that are at or near the distal loops of the DNA-binding arms (Williams and Maher III 2000). These residues are critical to HU function (Saitoh et al. 1999), and their Raman markers provide a convenient basis for probing HU/DNA recognition in nucleoprotein complexes. In addition, the deficiency of aromatic side-chains in HUBst, specifically the absence of both Trp and Tyr residues, facilitates the identification of Raman markers of key aliphatic side-chains. The abundant and largely conserved Ala residues of HUBst (Fig. 1), which function in -helix packing, also exhibit an identifiable Raman signature and a diagnostic pattern of alanyl peptide exchange. Because alanines of HU proteins have been correlated with protein thermostability (Wilson et al. 1990), the Raman procedures developed here are expected to be useful in assessing the roles of local alanyl environments in subunit stability and assembly. The present study reveals that eight alanyl sites in -helices 1 and 2 of the HUBst dimer are exchange protected in the native subunit fold, consistent with strong N-H...O hydrogen bonding of the peptides. It will be of interest to probe the dynamics and interactions of these Ala sites in the HUBst:DNA complex, as well as in other protein/DNA complexes.
Structural studies of several families of DNA-binding architectural factors, including the histone-like proteins HU and IHF, as well as HMG-box and winged-helix proteins, illustrate how modest differences among members of the same family determine specificity in the mechanism of DNA recognition (Gajiwala and Burley 2000; Travers 2000). Although HU and IHF have high sequence and structure homology, HU is distinguished from IHF by its ability to induce superhelical stress in covalently closed DNA duplexes (Ogata et al. 1997). The different outcomes of HU/DNA and IHF/DNA recognition may be distinguishable by sensitive Raman difference methods, which have the capacity to differentiate even modestly supercoiled DNA from relaxed DNA (Serban et al. 2002). The present findings represent a starting point for subsequent Raman and UVRR investigation of HU:DNA and IHF:DNA complexes and comparative analysis of their respective molecular mechanisms of interaction with topological variants of high-molecular-weight DNA. These studies also provide a basis for future application of Raman methods to other proteins containing alanyl and prolyl residues at inter-subunit and subunit/DNA interfaces.
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230 = 2.3 mL/mg/cm and 280 = 0.56 mL/mg/cm) were dialyzed against 100 mM Na phosphate, 100 mM NaCl, 0.1 mM PMSF, 0.1 mM EDTA (pH 7.5; buffer B). The protein was subsequently applied to a Bio-Scale S5 column (BioRad) and eluted with a linear gradient of 0.1 to 1.0 M Na phosphate in buffer B. The purified HUBst fractions were analyzed by SDS-PAGE and dialyzed against 20 mM Tris and 50 mM NaCl (pH 7.5). Finally, the protein was either concentrated in this buffer for use in unfolding/refolding experiments or exchanged with 100 mM NaCl solution and then concentrated using a Centricon-3 ultrafiltrator of 3,000-Da cutoff (Amicon, Inc.) for use in Raman analyses. The concentration of purified HUBst was determined by UV absorbance using a molar extinction coefficient at 258 nm of 258= 0.076 mL/mg/cm (1482 M-1 • cm-1; Welfle et al. 1992).
Deuterium exchange
Deuterium exchange of native HUBst was carried out by incubation in either 100 mM NaCl/D2O (at pD 7.9) or 10 mM Tris, 50 mM NaCl/D2O (pD 7.9) for 4 h at 20 °C. This procedure accomplishes H/D exchange of labile main-chain (peptide NH ND) and side-chain sites (e.g., Lys and Arg) not protected by the native fold. Complete H/D exchange of all labile sites was accomplished by incubating the unfolded protein for 1 h in 6 M guanidinium deuteriochloride (GuDCl). The fully exchanged protein was subsequently refolded by extensive dialysis against 10 mM Tris, 50 mM NaCl/D2O (pD 7.9) and concentrated on a Centricon-3 ultrafiltrator as required for Raman spectroscopy. Completion of H/D exchange was assessed by elimination of Raman intensity owing to NH and OH group hydrogenic stretching modes (3200 to 3400 cm-1). The integrity of the refolded protein was assessed by restoration of the CD profile associated with the native structure. The unfolding/refolding cycle using nondeuterated reagents was also shown to restore the Raman signature of the native structure. H/D exchanges of model proteins (bradykinin, PLA, and related amino acids) were carried out similarly.
Raman spectroscopy
Raman spectra of HUBst were obtained from H2O (D2O) solutions prepared at 20 µg/µL in either 0.1 M NaCl (pH 7.5, pD 7.9) or 10 mM Tris, 50 mM NaCl (pH 7.5, pD 7.9). Solutions of L-arginine and PLA were prepared at 5 mM and adjusted to pH 7.5 (pD 7.9) with HCl (DCl) or NaOH (NaOD). Aliquots of ~5 µL were sealed in glass capillaries (KIMAX No. 34507) and thermostated at 20 °C (Thomas, Jr., and Barylski 1970) for data collections. Spectra were excited at 532 nm by using a solid-state Nd:YVO4 laser (Verdi, Coherent, Inc.) and collected on a Spex 500 M single spectrograph (Instruments S.A.) equipped with a holographic notch filter and liquid nitrogen–cooled, back-thinned, charge-coupled-device (CCD) detector of 2000 x 800 pixels (Spectrum One, Instruments S.A.). The radiant power at the sample was ~30 mW. The effective spectral resolution was 3 cm-1. Raman frequencies are accurate to ±0.5 cm-1, as judged by calibration of the spectrophotometer using the 459.5 cm-1 band of CCl4. Spectra shown below are the accumulated averages of 10 to 30 exposures of 40 s each. Further details of the instrumentation and data collection protocols are given elsewhere (Movileanu et al. 1999).
UVRR spectroscopy
UVRR spectra were excited with 
1 mW of 229-nm radiation from an Innova 300 FreD argon laser (Coherent, Inc.) and were collected on a Spex model 750M single-grating (2400 g/mm) spectrograph equipped with a Spex model Spectrum-1 liquid nitrogen–cooled CCD detector (ISA). Spectrometer resolution (8 cm-1), wavenumber calibration (versus CCl3CN), and performance characteristics have been described (Russell et al. 1995). UVRR spectra were collected from H2O (D2O) solutions of HUBst prepared at 2 µg/µL in 10 mM Tris (pH 7.5, pD 7.9). Samples were maintained at room temperature (20 °C) in a rotating quartz cell (300 rpm).
The prominent UVRR band from phenylalanines of HUBst (1003 cm-1) also serves as an internal frequency and intensity standard. The reported spectral data represent averages obtained from five independent experiments, which exhibited band intensity deviations generally not exceeding ±5% and wavenumber deviations not exceeding ±1 cm-1 for sharp bands or ±4 cm-1 for broad or very weak bands. As in previous work (Russell et al. 1995; Wen et al. 1997), no photochemical damage was observed for any sample as judged by the absence of transient or irreversible changes in the UVRR spectra within the time of data collection (<10 min). Exposure thresholds for photochemical decomposition (>12 min) were established for each sample at the laser power used and were never exceeded. Other potential sources of error, such as polarization artifacts and sample self-absorption effects, are minimized by the experimental design and collection protocols, as detailed elsewhere (Russell et al. 1995; Wen et al. 1997; Wen and Thomas, Jr., 1998).
CD spectroscopy
CD spectra were recorded from HUBst solutions (~0.3 mg/mL in 1 mM sodium cacodylate buffer at pH 7.0, 20°C) using a Jasco J-720 spectropolarimeter (Japan Spectroscopic, Co.) and an optical path of 0.1 cm. The spectropolarimeter was calibrated using ammonium d-camphor-10-sulfonate. CD data referenced below represent the accumulated averages of five scans, each obtained at a scan rate of 0.5 nm/sec.
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References |
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