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A correction to the sequence of the alpha chains of horse haemoglobin
.I. Mol. BioZ. (1976), 103, 675-677
A Correction to the Sequence of the Alpha Chains of Horse Haemoglobin An atomic model of horse aquomethaemoglobin extensively refined against 2.0 f, X-ray data suggested that an Ala and a Gly residue in the published sequence of the Q chain were interchanged. Chemical analysis has confirmed our interpretation of the model.
The sequence of the CLchain of horse haemoglobin was determined by Braunitzer & Matsuda (1963). It shows Ala in position E12(63) and Gly at E14(65). The former Ala is homologous with other mammalian haemoglobins except that of rabbit, where it is replaced by Ser, but the Gly at El4 would be unique, all other mammals having Ala in this position (Dayhoff, 1972). The structure of horse methaemoglobin has recently been refined at 2.0 A resolution. The refinement showed features which have led us to re-examine the relevant part of the sequence and to conclude that it should be inverted with Gly at El2 and Ala at E14.
Crystallographic
Evidence
During the refinement of the atomic model of horse aquomethaemoglobin (metHb) against 2.0 A X-ray data (R. C. Ladner, E. G. Heidner & M. F. Perutz, manuscript in preparation) a short contact (<3.0 A) between the methyl carbon of Ala E12(63)a and the 0 of Ala B2(21)a and the N of Gly B3(22)a persisted through several cycles. We did not think that this was due to mispositioning of either helix, since both were quite well-resolved in our Fourier maps. We did find that real-space refinement of the Gaussian radius (Diamond, 1971) of the methyl of Ala El2 gave the largest value permitted (2.8 A, whereas well-resolved atoms fell between 1.3 A and 1.5 A) and that there was almost no density at the methyl in the Fourier maps. At this point we discussed the possibility of a sequencing error with J. V. Kilmartin, who assured us that he had isolated the fragment in question and that its composition agreed with that of the published sequence. When we examined an observed-calculated Fourier, however, we found a strong positive peak of 0.5 e/A3 adjacent to the Ca of Gly E14(65)a (see Fig. 1). An examination of the atomic model of metHb showed that conversion of “Gly” El4 to Ala would make a cap on the haem pocket. C/3 would lie in the haem plane and make van der Waals’ contacts of 4.1 A with the methyl of vinyl ring I, 4.5 A with the methine C between rings I and IV, and 4.0 A with the methyl C on ring IV. Furthermore, residue El4 in the p chains is always Ala (primates, mouse, rabbit and grey kangaroo), Thr (ruffed lemur), or Ser (dog, horse, cow, pig, llama, sheep and goat), but never Gly. Thus we proposed that the sequence should read El2 = Gly, El3 = Asp, El4 = Ala, which leaves the composition of the peptide unchanged. This brings El4 into line with other mammalian haemoglobins, but makes El2 unique in horse, since this position is generally occupied by Ala.
076
12. (‘.
I,.ll)NEH,
(:.
11.
.iIR
:\&‘I)
.I.
H.
FO(:(:
Fra. 1. The difference Fourier map had coefficients (1 P,,,j - j &“lcalcl) exp(i c(~& and is shown superimposed on part of the model for the a chain of horse aquomethaemoglobin. 1Fc:alcl and aoalc are the amplitude and phase calculated from a model which had Ala at residue E12a and Gly at residue E14a. The map is contoured starting at *0.20 e/A3 at intervals of 0.10 e/A3 with negative contours dashed. In addition to atomic positions, which were subject to constraints on bond distances and angles, we refined a Gaussian radius (equivalent to an isotropic temperature factor) per atom and an occupancy per residue. to the b axis and so that the two Cg atoms in question are in The sectioning planes are parallel the same plane; section 0 passes through the carbonyl C of residue El2 and 2 sections at 1 4 spacing are shown on either side of section 0. The 3 fragments of model shown are (from left to right) : (1) part of the B helix, His Bl (without its imidazole ring), Ala B2, and Gly B3; (2) part of the E helix, Val Ell, Gly El2 (bearing the false methyl group as a large open circle joinrd by a dashed bond), Asp E13, and Ala El4 (with the new methyl as an open circle); and (3) part of the CLhaem with rings I ant1 IV up, the Intt,et nearer the vieww, t,ho iron-linked water projects towards Val Ell.
Chemical Evidence The CI and /3 chains of horse globin were separated, and 20 nmol of the tryptic peptide crTp9 (residues 62 to 90) isolated as described by Kilmartin & Clegg (1967). The results of the first four steps of a manual dansyl-Edman sequential degradation by the method described by Air et al. (1975) are shown in Figure 2. The sequence is clearly 62 63 64 65 Val-Gly-Asx-Ala-, in which Ala (63) and Gly (65) of the published
sequence of horse a-globin
are reversed.
Our results confirm that a fully refined X-ray structure at 2-O A can localize a single atom under favourable conditions. Three other examples occurred in the ,!? chains: through clerical errors the sequence refined was Ala(lll)fi-Leu-Val-Valin place of the correct sequence Val(lll)/I-Val-Val-Leu-. The error at lllj3 was noticed by chance but could have been seen in an observed-calculated difference map, where two positive peaks appeared next. to the cl,3 of Ala. Similarly, the transposition between 112 and 114 was noticed by Dr T. Takano but could have been seen in the observedcalculated difference map: the C6 atoms of the false Leu faded out by generating a large radius and a second Cy appeared; the Cy atoms of the false Val had their radii constrained to be the same so that the false Cy could not fade out but it appeared
embedded in negative positive peaks beyond
difference density, the correct Cy.
Medical Research Council Cambridge, England I+wi\wl
27 Fc~hrrlar>-
Laboratory
while
the correct
of Molecular
Biology
CS atoms
emerged
as
ROBERT C. LADNER GILLIAN M. AIR *JANICE H. FOGG
1976 HEFERENCFS
1,
Air, G. M., Blackburn, E. H., Sanger, E’. & Coulson, A. R. (1975). J. Nol. Uiol. 96, 703 -719. Braunitzer, G. & Matsuda, G. (1963). J. Biochena. (Tokyo), 53, 262-263. Dayhoff, M. 0. (1972). Editor of Atlas of Protein Sequence and Structure, vol. 5. p. 13.66 ff. National Biomedical Research Foundation, Washington. Diamond, R. (1!)71). Acta Cry&.ullogr. sect. d, 27, 436-452. Kilrm~rtill, J. V. 8: Clegg, J. B. (l-967). Xattere (Ihdon), 213, 269 27 I.
A Correction to the Sequence of the Alpha Chains of Horse Haemoglobin An atomic model of horse aquomethaemoglobin extensively refined against 2.0 f, X-ray data suggested that an Ala and a Gly residue in the published sequence of the Q chain were interchanged. Chemical analysis has confirmed our interpretation of the model.
The sequence of the CLchain of horse haemoglobin was determined by Braunitzer & Matsuda (1963). It shows Ala in position E12(63) and Gly at E14(65). The former Ala is homologous with other mammalian haemoglobins except that of rabbit, where it is replaced by Ser, but the Gly at El4 would be unique, all other mammals having Ala in this position (Dayhoff, 1972). The structure of horse methaemoglobin has recently been refined at 2.0 A resolution. The refinement showed features which have led us to re-examine the relevant part of the sequence and to conclude that it should be inverted with Gly at El2 and Ala at E14.
Crystallographic
Evidence
During the refinement of the atomic model of horse aquomethaemoglobin (metHb) against 2.0 A X-ray data (R. C. Ladner, E. G. Heidner & M. F. Perutz, manuscript in preparation) a short contact (<3.0 A) between the methyl carbon of Ala E12(63)a and the 0 of Ala B2(21)a and the N of Gly B3(22)a persisted through several cycles. We did not think that this was due to mispositioning of either helix, since both were quite well-resolved in our Fourier maps. We did find that real-space refinement of the Gaussian radius (Diamond, 1971) of the methyl of Ala El2 gave the largest value permitted (2.8 A, whereas well-resolved atoms fell between 1.3 A and 1.5 A) and that there was almost no density at the methyl in the Fourier maps. At this point we discussed the possibility of a sequencing error with J. V. Kilmartin, who assured us that he had isolated the fragment in question and that its composition agreed with that of the published sequence. When we examined an observed-calculated Fourier, however, we found a strong positive peak of 0.5 e/A3 adjacent to the Ca of Gly E14(65)a (see Fig. 1). An examination of the atomic model of metHb showed that conversion of “Gly” El4 to Ala would make a cap on the haem pocket. C/3 would lie in the haem plane and make van der Waals’ contacts of 4.1 A with the methyl of vinyl ring I, 4.5 A with the methine C between rings I and IV, and 4.0 A with the methyl C on ring IV. Furthermore, residue El4 in the p chains is always Ala (primates, mouse, rabbit and grey kangaroo), Thr (ruffed lemur), or Ser (dog, horse, cow, pig, llama, sheep and goat), but never Gly. Thus we proposed that the sequence should read El2 = Gly, El3 = Asp, El4 = Ala, which leaves the composition of the peptide unchanged. This brings El4 into line with other mammalian haemoglobins, but makes El2 unique in horse, since this position is generally occupied by Ala.
076
12. (‘.
I,.ll)NEH,
(:.
11.
.iIR
:\&‘I)
.I.
H.
FO(:(:
Fra. 1. The difference Fourier map had coefficients (1 P,,,j - j &“lcalcl) exp(i c(~& and is shown superimposed on part of the model for the a chain of horse aquomethaemoglobin. 1Fc:alcl and aoalc are the amplitude and phase calculated from a model which had Ala at residue E12a and Gly at residue E14a. The map is contoured starting at *0.20 e/A3 at intervals of 0.10 e/A3 with negative contours dashed. In addition to atomic positions, which were subject to constraints on bond distances and angles, we refined a Gaussian radius (equivalent to an isotropic temperature factor) per atom and an occupancy per residue. to the b axis and so that the two Cg atoms in question are in The sectioning planes are parallel the same plane; section 0 passes through the carbonyl C of residue El2 and 2 sections at 1 4 spacing are shown on either side of section 0. The 3 fragments of model shown are (from left to right) : (1) part of the B helix, His Bl (without its imidazole ring), Ala B2, and Gly B3; (2) part of the E helix, Val Ell, Gly El2 (bearing the false methyl group as a large open circle joinrd by a dashed bond), Asp E13, and Ala El4 (with the new methyl as an open circle); and (3) part of the CLhaem with rings I ant1 IV up, the Intt,et nearer the vieww, t,ho iron-linked water projects towards Val Ell.
Chemical Evidence The CI and /3 chains of horse globin were separated, and 20 nmol of the tryptic peptide crTp9 (residues 62 to 90) isolated as described by Kilmartin & Clegg (1967). The results of the first four steps of a manual dansyl-Edman sequential degradation by the method described by Air et al. (1975) are shown in Figure 2. The sequence is clearly 62 63 64 65 Val-Gly-Asx-Ala-, in which Ala (63) and Gly (65) of the published
sequence of horse a-globin
are reversed.
Our results confirm that a fully refined X-ray structure at 2-O A can localize a single atom under favourable conditions. Three other examples occurred in the ,!? chains: through clerical errors the sequence refined was Ala(lll)fi-Leu-Val-Valin place of the correct sequence Val(lll)/I-Val-Val-Leu-. The error at lllj3 was noticed by chance but could have been seen in an observed-calculated difference map, where two positive peaks appeared next. to the cl,3 of Ala. Similarly, the transposition between 112 and 114 was noticed by Dr T. Takano but could have been seen in the observedcalculated difference map: the C6 atoms of the false Leu faded out by generating a large radius and a second Cy appeared; the Cy atoms of the false Val had their radii constrained to be the same so that the false Cy could not fade out but it appeared
embedded in negative positive peaks beyond
difference density, the correct Cy.
Medical Research Council Cambridge, England I+wi\wl
27 Fc~hrrlar>-
Laboratory
while
the correct
of Molecular
Biology
CS atoms
emerged
as
ROBERT C. LADNER GILLIAN M. AIR *JANICE H. FOGG
1976 HEFERENCFS
1,
Air, G. M., Blackburn, E. H., Sanger, E’. & Coulson, A. R. (1975). J. Nol. Uiol. 96, 703 -719. Braunitzer, G. & Matsuda, G. (1963). J. Biochena. (Tokyo), 53, 262-263. Dayhoff, M. 0. (1972). Editor of Atlas of Protein Sequence and Structure, vol. 5. p. 13.66 ff. National Biomedical Research Foundation, Washington. Diamond, R. (1!)71). Acta Cry&.ullogr. sect. d, 27, 436-452. Kilrm~rtill, J. V. 8: Clegg, J. B. (l-967). Xattere (Ihdon), 213, 269 27 I.