1 Department of Biochemistry and 3 Bioinformatics Centre, Bose Institute,P-1/12 CIT Scheme VIIM, Calcutta 700 054, India
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Abstract |
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Keywords: -helix/aromatic residues/interaction geometry/modelling/protein folding
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Introduction |
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Interaction between aromatic residues (Phe, Tyr, His and Trp) can contribute to the stability of the native fold (Burley and Petsko, 1985) and it has been suggested that aromatic clusters could be a determinant of thermal stability in thermophilic proteins (Kannan and Vishveshwara, 2000
). An aromatic residue has been implicated in the origin of DNA-binding specificity of Arc repressor (Schildbach et al., 1999
). For the proper understanding of the specificity of binding and stability, it is necessary to know if the aromatic rings have any preferred geometry of interaction and indeed the perpendicular (edge-to-face) orientation is generally favoured (Burley and Petsko, 1985
; Singh and Thornton, 1985
; Hunter et al., 1991
). However, none of these studies addressed the question of whether the geometry can vary depending on the relative location in a helix, if any particular pair is preferred among all the possible pairs of interacting aromatic residues and if within a pair a given residue has a preference to precede the other in the sequence.
Elucidation and quantification of different non-covalent interactions provide a foundation on which the modification of protein stability and the design of small proteins can be made in a rational way. It has been possible to dissect the contribution of local interactions to -helix stability using synthetic model peptides (Chakrabartty and Baldwin, 1995
; Serrano, 2000
). For example, the interaction of Phe8 with His12 has been shown to account for the unexpected stability of the C-peptide helix (the N-terminal 13 residues of RNase A) (Dadlez et al., 1988
; Shoemaker et al., 1990
; Armstrong et al., 1993
). Likewise, Trp and His at positions i and i + 4, respectively, can stabilize the helix by 1 kcal/mol when the histidine is protonated (Fernandez-Recio et al., 1997
). For the design of supersecondary structures containing
-helix, it is also imperative to know the interactions that straddle the helix terminus, linking residues in helix to those beyond (Aurora and Rose, 1998
). For a better understanding of the role of aromatic residues in providing stability to
-helices and their immediate neighbourhood, we analysed all known protein structures to identify pairs of interacting aromatic residues, both located within
-helices or one in a helix and the other within five residues of a helix-end, and then found the geometry of interaction. This led to the enumeration of patterns in sequence and structure involving the aromatic residues.
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Materials and methods |
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The -helices were located using the program DSSP (Kabsch and Sander, 1983
) and all the aromatic residues within helices and five residues from either end of the helices were identified; those with more than one conformation of the side chain were, however, excluded from further calculations. Distances between ring centroids of all aromatic residues in and around a given
-helix were calculated and any pair of residues within a distance of 7.5 Å [which was found to be optimal by Samanta et al. (Samanta et al., 1999
)] was assumed to be interacting. For the calculation of the geometry of interaction between the two rings, their centroids were calculated (for Trp, the mid-point of the CD2CE2 bond was taken as the centroid) and a molecular axes system defined (with the origin at the centroid of the aromatic residue which precedes in the sequence and the z-axis perpendicular to its ring plane). Two parameters were calculated, theinterplanar angle (P) and the angle (
) made by the z-axis with the line joining the origin to the centroid of the second ring.
The difference () in residue numbers between the two interacting residues, [Ari]1 and [Arj]2, where Ari/j stands for any of the four aromatic residues whose relative positions in the sequence are indicated by the subscript 1 or 2 and the numbers (Oi,j) of the various combinations of [Ari]1[Arj]2 pairs observed (for a given
) were found. The expected number (Ei,j) of the pairs is proportional to the product of the fractional abundances of the two residues in positions 1 and 2:
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The solvent accessible surface area (ASA) was computed using the program ACCESS (Hubbard, 1992), which is an implementation of the Lee and Richards (1971) algorithm. The relative accessibility of a residue (X) was obtained by dividing the observed ASA by its ASA in a model tripeptide, AlaXAla. Cartoon representations of molecules were generated using MOLSCRIPT (Kraulis, 1991
).
The codes for the PDB files used are given in the Appendix.
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Results and discussion |
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There are 2280 -helices in 434 protein chains considered, of which 1553 contain 0 or 1 aromatic residue and thus have no pairwise contact. The number of residues which are aromatic as a function of helix length is plotted in Figure 1
. The fitted straight line suggests that on average one aromatic residue is incorporated into the helix as its length increases by 10. This number is quite close to 11.4, the average percentage composition of aromatic residues in protein structures (Chakrabarti and Pal, 2001
). One feature of the plot, for which there is no obvious explanation, is the dip in the number of aromatic residues when the length is 9 and 18. If one looks at the percentages of helices with different numbers of aromatic residues (Figure 2
), one finds that the number of helices with no aromatic residue falls off monotonically as the length of the helix increases, but it is still possible to find long helices devoid of any aromatic residue. The numbers of helices with four or more aromatic residues show a reverse trend, appearing at a length of about 10. In between these two groups the percentages of helices with one or two aromatic residues remain fairly uniform over the helix length, being 3040% for the former (for helices up to a length of 20) and 2030% for the latter (when the helix is 920 residues long).
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Geometric considerations dictate that two helical residues can be in contact (centroid within 7.5 Å) only when there are specific sequence differences () between them. Table I
shows that 50% of all residues interact when they are located one or three residues apart, whereas a much greater percentage (94%) can interact when they are four residues apart. This is also shown by a shorter contact distance which is possible for the latter relative position. The average values of the closest distance between any two atoms of the interacting pair for different
values are: 4.1(6) Å for 1; 4.3(7) Å for 3 and 3.9 (6) Å for 4. Interestingly enough, even when the two residues are one helical turn away (
= 7 or 8) there are examples having interaction.
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As discussed by Samanta et al. (Samanta et al., 1999), the relative orientation of the second ring with respect to the first (in a pair) can be idealized at three sets of discrete values of the two angular parameters P and ñ (Figure 3
). Each relative orientation is designated by a two-letter code (ff, ot, ef, etc.). The first letter indicates if the first residue in a pair is interacting with its face (f), edge (e) or has the centroid of the second ring in an intermediate (offset or o) position. The second letter indicates if the second residue is tilted (t) with respect to the first or has its face (f ) or edge (e) pointing towards the first.
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It is of interest to know if any particular combination of aromatic residues is found to interact more than expected and data presented in Table III identify a few such pairs: HisHis when
= 1 and 4 and PheHis, TyrTrp and TrpPhe pairs when
= 3. The smallest (His) and the largest (Trp) aromatic residues are found as one of the interacting partners. Calculations on relative ordering of the two residues in a pair (see Materials and methods) also suggest that the PheHis pair has a much greater chance of occurrence than the HisPhe pair when
= 3 or 4 (particularly the latter).
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Using an alanine-based peptide system, Armstrong et al. have shown that the interaction between Phe and protonated His at relative position i / i + 4 (i.e. = 4) is helix stabilizing and is independent of the exact location of the pair in the helix (Armstrong et al., 1993
). Likewise, Fernandez-Recio et al. studied the helical content of several peptides in which a TrpHis pair was placed at i, i+3 or i, i + 4 in either the N to C or the C to N orientation and found that the TrpHis pair at i, i + 4 gives rise to the highest helical content (i.e. the most stabilizing) when the histidine is protonated (Fernandez-Recio et al., 1997
). However, none of these dealt with the geometry between the two rings. As discussed in the previous section, our analysis shows that the PheHis pair is observed significantly more than in the reverse order both when
=3 and 4. The geometry of His with respect to Phe is ot or oe (Figure 3
) when
= 3, similar to other pairs with the same sequence difference (Table II
). For the 12 cases observed with
= 4, the majority (seven) are in the geometry ft or ot (an additional two in of and the rest are scattered in other boxes of Figure 3
). With the tilted orientation of the His ring relative to Phe (when
= 4), an XH group (where X = C or N) usually at CD2 or ND1 of His is directed towards the
electron cloud of the Phe ring [with an average X···C distance of 3.7(5) Å] (Figure 6
) resulting in a CH···
or NH···
interaction (Mitchell et al., 1994
; Nishio et al., 1998
; Samanta et al., 2000
). When the His ring is protonated, the NH group at ND1 becomes available and the other CH protons in the ring carry more partial positive charge, resulting in the occurrence or the strengthening of the XH···
interaction (Samanta et al., 1998
). This explains the result from the solution studies showing an increase in the helical content of model peptides containing an aromaticHis pair (at i, i + 4) when His is protonated. Again, the reason why the order in which the two residues occur is important becomes apparent from the consideration of geometry. The two cases of HisPhe (i, i + 4) pair observed have the geometry et (using a different convention, where the geometry of the first ring, i.e. His is expressed relative to the second, i.e. Phe), so that the edge of Phe is pointed towards the face of the His ring in a tilted fashion, which is not particularly stabilizing. The stabilizing nature of the edge of His interacting with the face of an aromatic ring is also exemplified by a tertiary interaction between His18 and Trp 94 in barnase, where the protonation of His increases the stability of the protein by 1 kcal/mol (Loewenthal et al., 1992
). The geometry of His with respect to Trp (based on the PDB file, 1B20) is ft, as observed for most of the PheHis pairs (
= 4) in helix. When
= 3, the centroids of the two rings are further apart and an XH···
interaction appears to exist only for a few pairs with geometry ot.
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Interaction of a helical aromatic residue with another one beyond the helixhelix capping
Helix capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices and Aurora and Rose have systematically grouped the commonly observed motifs into seven classes, based on the positions of the two interacting hydrophobic residues across the helix termini (Aurora and Rose, 1998). As we enumerated all the interactions between an aromatic residue within a helix and another one within five residues from a helix end (Table IV
), we wanted to see if any of the aromaticaromatic pairs matched the patterns of hydrophobichydrophobic pairs. We could identify only two cases (when
= 5) with the already annotated capping motifs. Using the following nomenclature for helices and their flanking residues: ... N''N'NcapN1N2N3 ... C3C2C1CcapC'C'' ... (where N1 = HN and C1 = HC in Table IV
), the pattern with
= 5 at the helix N-terminus is equivalent to the N'
N4 motif of Aurora and Rose (Aurora and Rose, 1998
) here a Phe or Tyr residue one position prior to the Ncap interacts with a similar residue four positions after the Ncap. For about 37% of the 33 cases with
= 5 at the helix C-terminus the helix ends at X2 (i.e. C1 position) and these are equivalent to C''
C3 (or Schellman motif), with a Gly at the C' position. Interestingly, with two aromatic residues interacting, the proportion of Trp is fairly high, especially at the C'' position. For other values of
, the motifs identified here are unique and can be broadly described as an aromatic interaction involving a His occupying the capping position and an
-helix capped by a 310-helix.
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Like the His occupying the C1 position in an -helix interacting with a Phe ring four residues preceding it, a His at the Ccap position can interact in an analogous manner (geometry ot or ft), such that a CH or NH group of His is positioned over the face of the other aromatic ring. It can be seen in Table IV(b)
that when
= 4, His is found in large number at the Ccap position (with a Phe or Trp at C4). Interestingly, even at the other end (Ncap), His is found with the maximum number of occurrences when
= 3 [Table IV(a)
], and here too, the geometry is such that the edge of His can point towards the face of the partner aromatic ring. Even when
= 4, His is conspicuous as the first aromatic residue. Thus, although His is not favoured near the helix N-terminus (Richardson and Richardson, 1988
), in association with another aromatic residue following it by three or four residues, His can offer stability to a helical structure.
Residues in - and 310-helices
Short pieces of 310-helices occur fairly frequently in protein structures, especially at the termini of -helices (more common at the C-terminal end) (Richardson, 1981
; Baker and Hubbard, 1984
; Barlow and Thornton, 1988
; Doig et al., 1997
; Pal and Basu, 1999
). However, it is not clear what factors are responsible for the tightening up of the last
-helical turn into the 310 conformation. In this connection, it is interesting that aromatic residues (with a sequence difference of 3) from
- and 310-helices, which are contiguous along the chain, can interact [see
= 3 in Tables IV(b) and V
] and possibly stabilize the structure. The geometry is very nearly identical (Figure 7a
) with what has been observed when both the residues are in the same
-helix. One of the residues involved is usually Tyr (or Trp) with one of the intervening residues being hydrophilic. In 1YGE, there is a short hydrogen bond (2.73 Å) between the side chains of His and Tyr. Interestingly, there is an example (Figure 7b
) where there is a kink between the two types of helices caused by a Pro residue and two residues with
= 7 interact with each other, as has also been seen within a kinked
-helix (Figure 4e
).
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The existence of an interacting pair of aromatic residues located on two halves of a kinked helix (Figures 4e and 7b) made us look for a situation when such residues are from two different helices separated by a non-helical residue. The results presented in Table VI
reveal a few interesting points. When
= 7, the geometry is fixed with the edge of the first residue interacting with the face of the second. The angle between the two helix-axes is also fairly constant, being in the range 102130°, and the intervening residue does not have any secondary structure. When
= 8, however, the residue is in the turn conformation, the geometry is et and the angle is in the range 6686°. The near constancy of the interhelix angle and the geometry of packing of the aromatic residues against each other suggests that such aromatic pairs can act as a prop for maintaining the proper relative orientation of the helices and their interaction energy may be enough to force a residue out of what would otherwise have become a single helix into a non-helical conformation. Thus, the specific geometry of aromaticaromatic interaction can confer specificity (uniqueness) to the packing of secondary structural elements.
Interaction between aromatic residues from two sides of a helix
The possibility of aromatic residues located within five residues of the two helical ends was also considered. As expected, of the 27 cases the majority involve short helices. There are four cases with = 10 and the central helix of length 4, with the two aromatic residues being three and four residues away from the two ends of the helix (Figure 8
) in all the cases the geometry is ef and one of the residues is in another helix, while the other residue has a non-regular conformation. When residues from the two ends interact they are mostly (63%) Phe.
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Implications and summary |
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Aurora and Rose named capping motifs based on the closest pair of interacting hydrophobic residues that straddles the helix terminus (Aurora and Rose, 1998). We identified some new capping motifs (Table IV
) where a His is at the capping position. Although His is not found near the helix N-terminus (Richardson and Richardson, 1988
), this bias can be overcome and His can occupy the Ncap position in association with another aromatic residue following it by three or four positions (Table IV
). Hence there is scope for improvement of secondary structure prediction if information on pairwise structural correlations in
-helices, as observed here and also by others (Klingler and Brutlag, 1994
; Walther and Argos, 1996
), is used in conjunction with traditional secondary structural propensity values, which are derived assuming that amino acids have no effect on each other. Additionally, interactions in helices are assumed to be local confined to consecutive turns of the helix (Aurora et al., 1997
). Here we identified a few cases of aromatic residues which interact even though they are separated by a helical turn (Table VI
, Figures 4e and 7b
). Specificity of folding is generally ascribed to the presence of buried polar interactions (Lumb and Kim, 1995
). However, the near constancy of the geometry of the HisPhe interaction and the order in which the residues are to be placed within a helix for stability (Figure 6
) and the relative orientation between two helices when they have a pair of interacting aromatic residues (Table VI
) suggest that the specificity can also be controlled by aromaticaromatic interaction. Interacting aromatic pairs can be used for the design and modelling of proteins. Finally, the functional role of His is well known, this study also points to a structural role for the residue.
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Appendix |
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1A1IA, 1A1YI, 1A28B, 1A2PA, 1A2ZA, 1A34A, 1A3C_, 1A48_, 1A4IB, 1A6M_, 1A7S_, 1A8D_, 1A8E_, 1A9XB, 1ABA_, 1ADOA, 1ADS_, 1AE9B, 1AFWA, 1AGQA, 1AHO_, 1AIE_, 1ALVA, 1AMF_, 1AMM_, 1AMX_, 1AOCA, 1AOHB, 1APYA, 1AQB_, 1ARV_, 1ATLA, 1AUN_, 1AVWB, 1AXN_, 1AY7B, 1AYFA, 1AYL_, 1AYOA, 1AZO_, 1B0NA, 1B0NB, 1B0UA, 1B0YA, 1B16A, 1B2VA, 1B3AA, 1B4KB, 1B5EA, 1B65A, 1B67A, 1B6A_, 1B6G_, 1B7CA, 1B8OA, 1B93A, 1BA8A, 1BABB, 1BAM_, 1BBHA, 1BBPA, 1BDO_, 1BE9A, 1BEA_, 1BEC_, 1BENB, 1BF6A, 1BFG_, 1BFTA, 1BG6_, 1BGF_, 1BI5A, 1BJ7_, 1BK0_, 1BK7A, 1BKRA, 1BQCA, 1BRT_, 1BS4A, 1BS9_, 1BSMA, 1BTN_, 1BU7A, 1BX4A, 1BX7_, 1BXAA, 1BXOA, 1BY2_, 1BYI_, 1BYQA, 1BYRA, 1C24A, 1C2AA, 1C3D_, 1C3MA, 1C3WA, 1C52_, 1CBN_, 1CC8A, 1CCZA, 1CEQA, 1CEWI, 1CEX_, 1CF9A, 1CFB_, 1CG6A, 1CKAA, 1CLEA, 1CMBA, 1CNV_, 1COZA, 1CPO_, 1CPQ_, 1CQYA, 1CS1A, 1CTJ_, 1CTQA, 1CV8_, 1CVL_, 1CXQA, 1CXYA, 1CY5A, 1CYDA, 1CYO_, 1CZFA, 1CZPA, 1D3VA, 1D7PM, 1D9CB, 1DBWB, 1DCIA, 1DCS_, 1DF4A, 1DFNA, 1DG9A, 1DGWY, 1DHN_, 1DI6A, 1DIN_, 1DLFH, 1DLFL, 1DOKA, 1DOSA, 1DOZA, 1DPSD, 1DPTA, 1DUN_, 1DXGA, 1ECD_, 1ECPA, 1EDG_, 1EDMB, 1EGPA, 1EUS_, 1EXTB, 1EZM_, 1FCE_, 1FIPA, 1FIT_, 1FLEI, 1FLTV, 1FLTY, 1FNA_, 1FRPA, 1FUS_, 1FVKA, 1G3P_, 1GCI_, 1GDOB, 1GOF_, 1GP1A, 1GPEA, 1GSA_, 1GUQA, 1HFC_, 1HFES, 1HKA_, 1HLEB, 1HOE_, 1HPM_, 1HTRP, 1HUUA, 1HXN_, 1IAB_, 1ICFI, 1IDAA, 1IFC_, 1IIBA, 1ISUA, 1IXH_, 1JDW_, 1JER_, 1JHGA, 1KNB_, 1KOE_, 1KP6A, 1KPTA, 1KVEA, 1KVEB, 1LAM_, 1LATA, 1LBU_, 1LCL_, 1LKFA, 1LKKA, 1LOUA, 1LTSA, 1LTSC, 1LUCA, 1MAI_, 1MDC_, 1MFMA, 1MGTA, 1MKAA, 1MLA_, 1MML_, 1MOF_, 1MOLA, 1MOQ_, 1MPGA, 1MRJ_, 1MROA, 1MROB, 1MROC, 1MSI_, 1MSK_, 1MTYB, 1MTYG, 1MUGA, 1MUN_, 1NAR_, 1NBCA, 1NCOA, 1NIF_, 1NKD_, 1NKR_, 1NLS_, 1NOX_, 1NP4A, 1NPK_, 1NULB, 1OAA_, 1OBWA, 1OPD_, 1OPY_, 1ORC_, 1OTFA, 1PBE_, 1PCFA, 1PDO_, 1PGS_, 1PHF_, 1PLC_, 1PNE_, 1POA_, 1POC_, 1PPN_, 1PSRA, 1PTQ_, 1PTY_, 1PYMB, 1QB7A, 1QCXA, 1QCZA, 1QD1A, 1QDDA, 1QFMA, 1QFOA, 1QGIA, 1QGWB, 1QGWD, 1QH4A, 1QH5A, 1QH8A, 1QH8B, 1QHFA, 1QJ4A, 1QJ8A, 1QKSA, 1QMPD, 1QQ4A, 1QQ5A, 1QQP1, 1QQP2, 1QQP4, 1QREA, 1QRRA, 1QSGA, 1QTSA, 1QTWA, 1QU9A, 1RB9_, 1RCF_, 1REC_, 1REGY, 1RGEA, 1RHS_, 1RIE_, 1RZL_, 1SCJB, 1SFP_, 1SGPI, 1SLUA, 1SMD_, 1SMLA, 1SRA_, 1SUR_, 1SVFA, 1SVFB, 1SVPA, 1SVY_, 1SWUB, 1TAFA, 1TAXA, 1TC1A, 1TEN_, 1TGXA, 1TIB_, 1TIF_, 1TL2A, 1TML_, 1TOAA, 1TTBA, 1TVXB, 1U9AA, 1UBPA, 1UBPB, 1UNKA, 1UOX_, 1VCAA, 1VFRA, 1VFYA, 1VHH_, 1VID_, 1VIE_, 1VLS_, 1VNS_, 1VSRA, 1WAB_, 1WAPB, 1WDCA, 1WHI_, 1WHO_, 1WWCA, 1XNB_, 1YACA, 1YAGG, 1YCC_, 1YGE_, 1YTBA, 2A0B_, 2ABK_, 2ACY_, 2AHJC, 2ARCB, 2AYH_, 2BC2A, 2BOPA, 2BOSA, 2CBP_, 2CCYA, 2CHSA, 2CPGA, 2CTC_, 2DRI_, 2DTR_, 2EBN_, 2EBOA, 2END_, 2ERL_, 2FDN_, 2GAR_, 2GDM_, 2HBG_, 2HDDB, 2HFT_, 2HMZA, 2IGD_, 2ILK_, 2KNT_, 2LISA, 2MSBB, 2MYR_, 2NLRA, 2PII_, 2PSPA, 2PTH_, 2PVBA, 2QWC_, 2RN2_, 2SAK_, 2SICI, 2SN3_, 2SNS_, 2SPCA, 2TNFA, 2TPSA, 2TRXA, 2TYSB, 2UBPC, 3CHBD, 3CHY_, 3CLA_, 3CYR_, 3ENG_, 3EZMA, 3GRS_, 3LZT_, 3PTE_, 3PVIA, 3PYP_, 3SDHA, 3SEB_, 3SIL_, 3STDA, 3TDT_, 3TSS_, 3VUB_, 4EUGA, 4MT2_, 5HPGA, 5PTI_, 6CEL_, 6GSVA, 7A3HA, 7RSA_, 8ABP_, 8PRKA, 9WGAA, 16PK_, 19HCA, 153L_, 256BA, 451C_.
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Notes |
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4 To whom correspondence should be addressed. E-mail: pinak{at}boseinst.ernet.in
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Acknowledgments |
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Received March 5, 2001; revised October 15, 2001; accepted November 11, 2001.