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1 Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892, USA
2 Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA
3 Chemical Physics Program, University of Maryland, College Park, Maryland 20742-2431, USA
Reprint requests to: Nico Tjandra, Laboratory of Biophysical Chemistry, Bldg. 50, Room 3503, NHLBI, NIH, Bethesda, MD 20892, USA; e-mail: nico{at}helix.nih.gov; fax: (301) 402-3405; or Carol Carter, Department of Molecular Genetics and Microbiology, Life Sciences Bldg., SUNY at Stony Brook, Stony Brook, NY 11794-5222, USA; e-mail: ccarter{at}ms.cc.sunysb.edu; fax: (631) 632-9797.
(RECEIVED October 10, 2002; FINAL REVISION January 15, 2003; ACCEPTED January 17, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0235903.
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Abstract |
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, and NOE) have been used to characterize the backbone dynamics of the N-terminal domain (NTD) of the oncovirus HTLV-I and to compare with the CA NTD of HIV-1. Large variations in the 15N heteronuclear NOEs and transversal relaxation rates for individual residues are consistent with the bundle RMSD of the previously calculated NMR structures. The ß-hairpin and CyP-A loop exhibit different mobility in HTLV-I and HIV-1. The overall hydrodynamic property of the HTLV-I capsid NTD is quite distinct from the HIV-1. Keywords: HTLV-I; capsid protein; retrovirus; NMR spectroscopy; relaxation; mutant; CyP-A
Abbreviations: HTLV-I, human T-cell leukemia virus type-I • HIV-1, human immunodeficiency virus type-I • CA, capsid • NTD, N-terminal domain • CyP-A, cyclophilin-A • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser effect • RSV, Rous sarcoma virus • MLV, murine leukemia virus • T1, spin-lattice relaxation time • T1, spin-spin relaxation time • CPMG, Carr-Purcell-Meiboom-Gill • SD, standard deviation • psec, picosecond • 
c, overall correlation time • S2, order parameter
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Introduction |
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In all mature retrovirus particles, the CA protein forms the shell of an inner core structure that encases the diploid RNA genome and the replicative enzymes. Whereas the noninfectious, immature particles look alike in the electron microscope, the mature particles are morphologically distinct and are characterized by the shape of the capsid core structure. The mature virion of HIV-1 and other lentiviruses is conical, spherical for HTLV-I, and an irregular polyhedron for Rous Sarcoma Virus (RSV) and Murine Leukemia Virus (MLV). The N-terminal ß-hairpin orientation may contribute to the various condensed core assemblages, as the CA NTD undergoes refolding after proteolysis from a structural precursor polyprotein (Gag), and this event influences capsid assembly (von Schwedler et al. 1998; Tang et al. 2002). Supporting this notion, we found that the conserved Asp residue that defines the orientation of the ß-hairpin (by forming the salt bridge with residue Pro 1) contacted the helical core such that there was an ~120° difference in the ß-hairpin orientation in the HTLV-I and HIV-1 CA NTDs. However, this structural difference alone cannot be attributed with certainty to the differences in the conical versus spherical core assemblages. The flexibility of this segment may also be a factor. So far, no comparisons of the dynamic properties of these two capsids have been carried out.
The HIV-1 CA domain binds human cyclophilin-A (CyP-A; Luban et al. 1993). The binding site is on a flexible, proline-rich loop situated between the least conserved region of the CA (between the fourth and the last helices of the core) showing variations in length and orientation among prototypes of the retroviral subgroups (HTLV-I, HIV-1, RSV, EIAV). Although the Ala-Gly-Pro-Leu/Ile motif in this loop of HTLV-I appears to be more exposed in the CA134 structure than in the 16-residue-deleted structure CA16–214 (Khorasanizadeh et al. 1999), we found that the HTLV-I CA does not bind CyP-A. We suggested that the length of the CyP-A-binding loop and the slow cis–trans transitions of residue Pro 90 in HIV-1 might be more important factors in the CyP-A-binding activity. The possible contributions to functional differences in the CA proteins from flexibility of the backbone and/or side chains may provide additional insight into the CA assembling mechanism.
In this study, we performed quantitative 15N NMR relaxation measurements together with NOE build-up rate measurements to assess the HTLV-I CA NTD backbone dynamic properties. A similar study on HIV-1 CA concluded that several regions including the ß-hairpin and CyP-A-binding loop exhibit a high degree of conformational mobility. A comparison of our results with previous findings reported for HIV-1 revealed differences in their backbone dynamic profile that might be important in understanding the differences in assembly of their mature particles.
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Results |
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, and the steady-state 15N-[1H] nuclear Overhauser effect (NOE), were obtained for 99 of the 121 protonated backbone 15N nuclei by analysis of the two-dimensional 1H–15N correlation spectra. Data for residues Gly 7, Met 17, Val 67, Gln 115, Ser 127, and Asp 130 could not be fitted satisfactorily because of either significant overlapping or signal broadening. Residues 1, 5, 9, 10, 14, 32, 35, 50, 92, 96, 104, 125, and 131 are prolines.
The experimental values of T1, T1, and NOE are plotted versus the residue number in Figure 1
. The value of T1 for the rigid part of the molecule (residues 1–125) does not change dramatically with the sequence position and has an average value of 707.5 (±11.3) msec. The averages of the T1 values for residues 1 to 125, which are well defined in the solution structure, are 91.8 (±1.8) msec. Statistically significant larger T1 values were observed for residues 126 to 134 in the highly disordered C-terminal region, indicating that this region is more flexible than the rest of the protein on the picosecond/nanosecond time scale. Residues Gln 29 and Thr 88 have T1 values smaller than the average. The average backbone 15N T1 value of 80 msec is consistent with the size of monomeric HTLV-I in solution. All our samples containing 0.3, 0.6, and 0.8 mM protein exhibited similar linewidths, and the T1 values measured for these samples are indistinguishable. Therefore, we can conclude that the NTD of HTLV-I CA protein is monomeric in solution. In the HIV-1 case, Gitti et al. (1996) observed that concentrations higher than 1.3 mM result in broader resonances, indicating oligomerization.
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, indicating increased flexibility for this region on the picosecond/nanosecond time scale.
Internal motion analysis
For proteins the size of CA134, NOEs are very sensitive to fluctuations in the internal dynamics of the individual NH vector as well as the overall rotational diffusion of the molecule. NOE values exceeding the maximum theoretical value show highly restricted internal motion, whereas NOE values smaller than 0.65 indicate significant internal motion. The averages of the NOE values at the C terminus (residues 126–134) are 0.02, indicating rapid internal motion for the C-terminus tail. The averages of the NOE values for the secondary structure elements are ß1 = 0.80 ± 0.04, ß2 = 0.75 ± 0.02, 
1 = 0.75 ± 0.07, 2 = 0.73 ± 0.14, 3 = 0.79 ± 0.06, 4 = 0.76 ± 0.07, 5 = 0.77 ± 0.06, and 6 = 0.79 ± 0.04. The NOE values’ averages for the loops are 0.36 ± 0.62 for residues 5–9, 0.82 ± 0.17 for residues 13–16, 0.81 ± 0.14 for residues 31–34, 0.76 for residues 50–51, 0.79 ± 0.03 for residues 63–64, 0.71 ± 0.07 for residues 86–96, 0.71 ± 0.08 for residues 103–106, and 0.18 ± 0.46 for residues 123–134. The loop that connects ß1 to ß2 (residues 5–9) exhibits intermediate values and the C terminus (residues 123–134) the smallest values. The NOE values for residues from the CyP-A-binding loop (residues 86–96) are slightly lower, but not significant, than the average.
The shape of CA134 (49 x 47 x 36 Å) indicates that the molecule is nonspherical. The relative lengths of the principal axes of the inertia tensor for CA134 are 1.00:0.85:0.56. This implies that the rotational diffusion tensor is either axially symmetric or fully anisotropic. For anisotropic diffusion, the 15N T1/T2 ratio depends on the orientation of the NH bond vector relative to the rotational diffusion tensor, D (Woessner 1962). T1/T2 ratios are maximum when the NH bond vectors are parallel to the long axis of D and minimum when the NH bond vectors are perpendicular to the long axis. The 15N T1/T2 ratio is, to a first approximation, independent of the rapid internal motions and magnitude of the chemical shift anisotropy. The presence of very rapid internal motions causes almost the same fractional increase in T1 and T2. Thus, T1/T2 ratios can be used to derive the rotational diffusion tensor. One must make sure that residues with a significant contribution from internal motion to the observed T1/T2 ratios are eliminated. First, residues with NOE < 0.65 must be excluded because their change in T1 values is much bigger than the T2 variation. Second, residues that exhibit conformational exchange on a microsecond or millisecond time scale will shorten the T2 values. Such residues are recognized by a shorter T2 without a simultaneous increase of T1. To exclude the residues with slower internal motions, for any residue n the following criteria have been imposed:
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where 
T1
and T2 are the averages over the residues that have NOE > 0.65 and SD is the standard deviation of the T2. The residues that do not fulfill these criteria experience additional line broadening, commonly described by the exchange term Rex (Clore et al. 1990). Using the above criteria, Gln 29 and Thr 88 have been excluded from the fittings.
The NH bond vectors of an -helix are nearly parallel to the helix axis and their T1/T2 ratios are uniform. Different helices exhibit different average T1/T2 values corresponding to the orientation relative to the rotational diffusion tensor (Fig. 2). The observed average T1/T2 values for the different helices in CA134 have characteristic values of 1 = 7.78 ± 0.65, 2 = 8.46 ± 0.30, 3 = 8.15 ± 0.38, 4 = 7.58 ± 0.28, 5 = 7.50 ± 0.20, and 6 = 8.50 ± 0.38, whereas average values of 7.91 ± 0.60 and 7.59 ± 0.02 were obtained for the ß1 and ß2 strands, respectively. For a given molecule, assuming a uniform hydration shell, the diffusion tensor should be collinear with the inertia tensor (the axis of fastest diffusion, Dz, corresponds to that of minimal inertia). The actual angles between helix 6, the closest to the orientation of the minor principal axis of the inertia tensor, and the helix axes of helices 1 through 5 are 40°, 21°, 19°, 44°, and 59°, respectively. The average T1/T2 values of the six -helices correlate well with their orientation relative to the diffusion tensor.
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for resonances Ala 8, Arg 42, Gln 105, and Lys 129. Figure 3B shows the differences between R2disp(cp) obtained for cp values of 10 and 0.5 msec. The difference 
R2disp(cp) does not change dramatically with the sequence position. We conclude that the NTD of HTLV-I CA protein does not exhibit a high degree of conformational mobility on the millisecond/second time scale.
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and 
that describe the orientation of Dz, and the 
angle that defines the orientation of Dy, where Dz > Dy > Dx. As previously described by Tjandra et al. (1996), the above parameters are obtained by minimizing the difference, E, between the observed and the predicted T1/T2 ratios:
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where the summation is over all residues used in the fitting and is the estimated error in the measured T1/T2 ratio. The principal components of the diffusion tensor (Dx, Dy, and Dz) are uniquely defined by the correlation time, c = [2Tr(D)]-1, anisotropy D||/D
(with D = [Dx + Dy]/2 and D|| = Dz) and asymmetry Dx/Dy.
To estimate the correlation time, c = (6Dz)-1, the isotropic model, Dx = Dy = Dz, was first assumed. This one-dimensional Powell minimization generated a c = 8.43 nsec and a normalized error function E/N = 7.7. Second, the data were fitted into the axially symmetric model, Dx/Dy = 1, resulting in a significant reduction of the normalized error function relative to the isotropic model. The use of the fully asymmetric model, six-parameter fit, results in a further reduction of E/N. The results of the fitting to different rotational diffusion models are presented in Table 1.
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The principal components of the axially symmetric diffusion tensor are Dz = 2.34 x 107 sec-1 and Dx = Dy = 1.88 x 107 sec-1. The symmetry axis of the diffusion tensor is oriented within 30° of the principal axis of the inertia tensor.
Internal motion parameters S2 and e were obtained by minimizing the difference between the calculated and measured T1, T2, and NOE for each residue (Tjandra et al. 1996). During this minimization the global diffusion parameters were fixed to the statistically significant values found from the T1/T2 optimization. The results are shown in Figure 4A,B
. The square of generalized order parameter (S2) has average values of 0.85 for the whole molecule, neglecting the C-terminal region. In the cone model (Brainard and Szabo 1981), this would correspond to motion within a semiangle of 18°. The following residues exhibit lower S2 values: 0.73 for Trp 15, 0.72 for Gly 95, 0.71 for Gly 126, 0.59 for Ala 128, 0.45 for Lys 129, 0.46 for Trp 133, and 0.23 for Ala 134. With the cone model of Brainard and Szabo (1981), these values correspond to motion of the NH bond vectors in cones of semiangles 26°, 26°, 27°, 33°, 39°, 42°, 42°, and 53°, respectively. Effective correlation time (e) values for internal motions are below 100 psec and range as far as 210 psec for residue Phe 37. The sites with higher e values also have lower S2 values, with the exception of residues Ser 34 and Leu 97, which exhibit high e (160 and 110 psec, respectively) and S2 values within the average (0.90 and 0.87, respectively). The values of order parameters are completely independent of secondary structure.
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c, of 8.2 nsec, and the molecule tumbles with an axially symmetric diffusion tensor (D||/D = 1.25).
A comparison between the diffusion tensor’s principal axes of HTLV-I and HIV-1 is presented in Figure 5. The structural studies of the N-terminal domain of HIV-1 CA protein (Campos-Olivas and Summers 1999) have concluded that its highly asymmetric shape correlates well with its inertial tensor principal axes (of relative magnitudes of 1.00:0.76:0.46), which, in turn, correlate with the diffusion tensor such that the axis of the fastest diffusion is nearly collinear with the minimal inertia (they form a 22° angle). In HIV-1, the CyP-A-binding loop contributes to the magnitude of the largest axis of the diffusion tensor by pointing outward along that axis. In HTLV-I, the same loop is shorter and folded toward the helical cluster, therefore shortening the respective axis of the diffusion tensor. Moreover, helix 4 is tilted outward along the intermediate diffusion axis, bringing its magnitude closer to the "largest" axis. The shortest axis is slightly extended relative to the HIV’s value, owing to the ß-hairpin pointing out from the helical core. The relative ratio of the inertia tensor’s components for HTLV-I is 1.00:0.85:0.56. The diffusion tensor calculations correspond to an axially symmetric model, as described in the Results section, that correlates with the shape of the molecule and with the inertia tensor. The angle between the largest axis of the inertia tensor and the axis of the fastest diffusion is roughly 30°, whereas for the HIV-1, this angle is 22°. These angles might at first seem large; however, earlier comparisons made between the diffusion tensor calculated from NMR relaxation data and extensive hydrodynamic computations indicated that a deviation of ~12° between the two tensors can be expected (Tjandra et al. 1995, 1996). These studies also pointed out that the deviation of the NMR relaxation-derived diffusion tensor from the inertia tensor was even larger. Relatively high-resolution X-ray structures were used in these analyses; thus, imperfect local geometry or "structural noise" was not a big factor and their results therefore provided the lower limit for the angular deviation between these tensors. Note that small changes in the local geometry involving the relaxation dipoles (NH bonds) can change the overall orientation of the tensor dramatically. Even though we did not find any consistent "outlier" data point, we still cannot completely rule out a contribution from structural noise in our analysis. Furthermore, we used HYDRONMR (de la Torre et al. 2000) to try to estimate from the bead method what the expected diffusion tensor would be for these capsid proteins. Interestingly, the results of these calculations consistently showed the intermediate inertia axis to be the fastest diffusion axis for both HTLV-I and HIV-1 NTD capsids. This is in disagreement with both our result and a previously published dynamic analysis of the HIV-1 capsid (Campos-Olivas and Summers 1999).
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, the helices 2 and 6 have the largest average values of the T1/T2 ratios, and their helix axes are the most aligned with the axis of fastest diffusion. In contrast, the ß1 and ß2 strands have the lowest average T1/T2 values with the exception of the disordered C-terminal region. This indicates that the NH bond vectors in the ß-hairpin must be perpendicular to the principal axis of diffusion.
The orientation of the ß-hairpin in CA134 (away from the helical core) was consistent with all our generated NMR structures. This is mostly caused by the NH and CH measured dipolar couplings that define accurately the relative orientations of protein fragments to the molecular alignment frame and, therefore, to one another. If true, this hairpin’s fixed orientation would imply a reduced mobility on both slow and fast time scales. The dynamic measurements T1 and NOE have confirmed the rigidity of this structural element with values virtually identical to the rest of the well-structured protein backbone, in contrast with the obvious pattern of local mobility (on a fast time scale) exhibited by the ß-hairpin in HIV-1 in the same type of experiments (Campos-Olivas and Summers 1999). Mutation, deletion, and addition studies of the ß-hairpin region of HIV-1 capsid have shown that this region is essential for capsid assembly (Campbell and Vogt 1995; Gross et al. 1998, 2000; von Schwedler et al. 1998). Changes in this region can alter the shape of the mature particles. This region has been hypothesized to form the capsid–capsid interface in the mature viral particle. Our study revealed that differences in backbone dynamics of the ß-hairpin of retroviruses might also govern their respective capsid assembly by modulating the overall flexibility of the capsid–capsid interface.
Packaging of host CyP-A is important for HIV-1 infectivity. The real function of CyP-A in the context of HIV-1 infectivity is still not known. Several recent results indicated that CyP-A might facilitate capsid particle disassembly (Rothman and Schmid 1986; Thali et al. 1994; Braaten et al. 1996; Ackerson et al. 1998). Although the binding sites in both proteins are exposed and oriented away from the main core, only HIV-1 capsid binds CyP-A. The loops between helices 4 and 5 (6 in HIV-1), which contain the Ala-Gly-Pro-Leu/Ile motif for binding to the human CyP-A, differ in length, number of helices, and proline isomerization state (Cornilescu et al. 2001). The CyP-A loop in HIV-1 exhibits high mobility (Campos-Olivas and Summers 1999), whereas the same loop in HTLV-I shows less flexibility. This is another distinguishing feature between these loops in the two proteins. Thus the high degree of conformational mobility as well as the proposed Pro 90 cis- and trans-isomerization may facilitate CyP-A binding to the HIV-1 CA. This, in turn, may differentiate how the core particles are disassembled in these two retroviruses.
In summary, our results identify clear differences in the backbone dynamic properties of the HTLV-I CA NTD, as compared with the HIV-1 structure. The fact that formation of at least one region (the N-terminal ß-hairpin) is a critical step in the viral capsid maturation process indicates that the flexibility of the elements that comprise the structure is also likely to be an important determinant of correct core assembly. The mobility of the loop containing the CyP-A-binding motif is different in the two capsid proteins. Perhaps a more flexible loop, such as in the HIV-1 capsid, is needed to allow the cis–trans-isomerization of the proline in this region, thus promoting CyP-A interaction. The difference in the hydrodynamic shapes between the HTLV-I and HIV-1 capsids that is reflected in their varying diffusion tensors must play an important role in their quaternary structures. Based on the hard-sphere interaction argument alone, one can already see that the packing of these proteins to assemble their associated core particles must be different. Clearly, having access to both local backbone dynamic and global hydrodynamic properties of retroviral capsid proteins is crucial in understanding the molecular basis of their functions.
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Materials and methods |
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NMR data collection
All NMR experiments were carried out at 27°C on Bruker AVANCE 600, operating at the 1H frequency of 600 MHz, equipped with a 5-mm shielded triple-gradient triple resonance probe. The 250-µL NMR sample contained 0.8 mM uniformly 15N-labeled protein in 92.5% H2O/7.5% 2H2O and 10 mM Tris-acetate, 0.1 mM NaN3 (pH 6.0). The 1H was positioned at the water frequency (4.78 ppm), and the 15N carrier was at 116.50 ppm for all experiments. The spectra were processed using the NMRPipe package (Delaglio et al. 1995) and analyzed with PIPP/STAPP (Garrett et al. 1991). The indirect dimension data were zero-filled prior to Fourier transformation.
Backbone 15N relaxation measurements
Spin–lattice (T1) and spin–spin (T1) relaxation times for the backbone 15N nuclei were measured using conventional pulse sequences (Barbato et al. 1992), adapted to include the Watergate scheme (Piotto et al. 1992), pulsed field gradient (Bax and Pochapsky 1992), and a semiconstant time evolution period in t1 (Grzesiek and Bax 1993a). The T1 experiment used a continuous 15N spin-lock field strength of 2.5 kHz (Peng et al. 1991).
All experiments were acquired as 512*(t2) x 128*(t1) data sets with 64, 96, and 128 scans per t1 point for T1, T1, and NOE, respectively. The T1 relaxation decay was sampled at eight different time points: 12, 84, 204, 324, 404, 564, 804, and 1004 msec. For the T1 data, eight spectra were recorded in an interleaved manner (to minimize any effects of spectrometer drift and sample heating) using the following 15N relaxation delays: 4.9, 14.5, 26.5, 38.5, 60.1, 88.9, 112.9, and 139.3 msec. 15N-[1H]-NOE values were measured using the water flip-back NOE pulse sequence described by Grzesiek and Bax (1993b) with a relaxation delay of 3.8 sec.
All T1, T1, and NOE experiments were performed twice, more than 1 wk apart, to calculate the pairwise root mean square difference (RMSD). The resulting random errors (half the pairwise RMSD values) used in the calculations were: 1.6% (T1), 2.0% (T2), and 7.8% (NOE). Relaxation times were calculated by nonlinear fittings of the delay-dependent peak intensities to an exponential decay function with a peak intensity baseline of zero.
15N-[1H]-NOE values were calculated as the intensity ratios of the 15N–1H correlation peaks from pairs of spectra acquired with and without 1H presaturation during the recycle time. The saturated and unsaturated experiments were interleaved; the absence of saturation was obtained by shifting the proton frequency off-resonance by ~3 MHz during the recycle time; 128 scans per t1 point were accumulated. 15N-[1H]-NOE values were corrected as previously described to compensate for the effects of incomplete 1H magnetization recovery (Grzesiek and Bax 1993b).
Relaxation dispersion data were obtained using a modified proton-detected 15N Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence (Loria et al. 1999) performed in a constant time (CT) manner (Tollinger et al. 2001) with a CT delay of 80 msec. Each 2D spectrum was acquired as 512*(t2) x 128*(t1) data sets with 64 scans per t1 point. Relaxation decay curves were measured for nine values of cp: 0.5, 0.67, 1, 1.25, 2, 2.5, 3.33, 5, and 10 msec, where cp is the delay between pulses in the spin-echo pulse train.
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Acknowledgments |
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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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), Gln 105 (•), and Lys 129 (
