A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

Biochimica et Biophysica Acta 914 (1987) 40-48 Elsevier

40

BBA32882

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin Irina M. Russu, Nancy T. Ho and Chien Ho Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA (U.S.A.) (Received 10 December 1986)

Key words: Hemoglobin, adult; Quaternary structure; Subunit interface; NMR; Nuclear Overhauser effect; (Human)

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated a or fl subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfieid from H20 to the hydrogen-bonded proton between al03(G10)His and fll08(G10)Asn at the a l f l 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H20 previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformationai effects of single amino-acid substitutions in mutant hemoglobin molecules.

Introduction Proton nuclear magnetic resonance (NMR) spectroscopy has emerged in the last two decades as a unique tool to investigate structure-function relationships in human normal and abnormal he-

Abbreviations: NMR, nuclear magnetic resonance; Hb, hemoglobin; Hb A, human normal adult hemoglobin; NOE, nuclear Overhauser effect; DSS, 2,2-dimethyl-2-silapentane-5sulfonate. This work was presented in part at the 30th Annual Meeting of the Biophysical Society, February 9-13, 1986, San Francisco, CA, U.S.A. Correspondence: C. Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.

moglobins (Hb). The power of this technique arises from the large number of amino-acid residues of Hb whose conformations and dynamics can be individually monitored and characterized by NMR (for recent reviews, see Refs. 1 and 2). Of special interest for understanding the structure-function relationship in Hb are those amino-acid residues which are situated at the intersubunit interfaces of the Hb tetramer and are involved in the hydrogen-bonding interactions responsible for the quaternary structures of deoxy and ligated Hb. Several of these amino-acid residues give rise to exchangeable proton NMR resonances over the spectral region 5.0-10.0 ppm downfield from the water proton resonance. Using appropriate mutant hemoglobins, several of these low-field exchangeable proton resonances of human normal adult hemoglobin (Hb A) in both deoxy and ligated

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

41 forms have been assigned by this laboratory to specific amino-acid residues [3-5]. Previous work from this laboratory has also demonstrated that these exchangeable proton resonances of Hb A provide excellent probes for correlating the binding of ligand to individual a a n d / 3 chains of Hb A and the quarternary structure transitions responsible for the cooperativity in ligand binding [6,7]. More recently, these exchangeable proton resonances of Hb have also been used to assess the long-range effects of single-site structural perturbations upon the conformation and dynamics of the Hb molecule [2]. In the present work, we have extended these N M R studies by an investigation of the nuclear Overhauser effect (NOE) of the inter-subunit low-field exchangeable proton resonances of deoxy and ligated Hb A. By using the initial NOE build-up rates, we have observed specific NOEs for each of the exchangeable proton resonances investigated. These NOEs allow us to monitor additional protons of the amino-acid residues situated at the al/3~ and %f12 interfaces of the Hb molecule. Thus, they provide new probes for the conformations and dynamics at the subunit interfaces in Hb. The NOEs observed in the present work also confirm the assignments of the exchangeable proton resonances of deoxy Hb A at + 8.2 ( + 8.1 ppm in ligated Hb) and + 9.4 ppm previously proposed by this laboratory [3,4]. Moreover, they allow us to assign the exchangeable proton resonance at + 7.5 ppm in deoxy Hb A ( + 7 . 4 pp, in HbCO A) to the intersubunit H-bond between al03(G10)His and/3108(G10)Asn at the al/3~ interface.

Kilmartin et al. [10,11] and modified by our laboratory as described [7].

Methods The coordinates of protons were generated starting from the X-ray diffraction coordinates of deoxy Hb A (symmetry averaged, Fermi, G. and Perutz, M.F.) and HbCO A ( N R G refined, Baldwin, J.) available from the Protein Data Bank at Brookhaven National Laboratory. For each amino-acid residue, the energy-refined standard geometry was assumed [12]. For protons involved in H-bonds, the coordinates were further adjusted according to average H-bond lengths and angles [131. The N M R measurements were performed on a Bruker WH-300 MHz spectrometer using a 16-bit digitizer. The water proton resonance was suppressed using the 'jump and return' pulse sequence introduced by Plateau and Gueron. To confirm that the proton resonances of interest were exchangeable, control experiments were carried out for Hb solutions in fully deuterated buffer as described previously [1]. The NOE experiments were carried out using the truncated-driven NOE pulse sequence proposed by Wagner and Wfithrich [15]: ( - t 1 ( ~ ) - o b s e r v a t i o n - t 2 - ) , . The low-power radio-frequency field was applied over the entire time interval tl, which ranged from 0.1 to 0.05 s.

a2

7.5

9.4

5.4

78 Zl

59 5.9

8.1

5.3 4.9

57

Materials

5.5 Z4

+~

5.0

5,4

8.1

+,b

4.9

6.4

Deoxy HbA

Experimental

Hb A was prepared from normal human blood obtained from the local blood bank by the procedure of Drabkin [8]. All the Hb samples used for the N M R experiments were of 2 mM concentration (Hb tetramer) in 0.1 M phosphate buffer at p H 7.0 with 5% 2H20 for the locking signal. The deoxygenation of the Hb samples was carried out as described previously [9]. The isolated a and fl chains were prepared according to the procedures introduced by

4.5

5.2

5.9 5.7

÷;

÷~

4

" 48

÷;

PPM from Hz0

Fig. 1. 300 MHz I H - N M R spectra of Hb A in 0.1 M phosphate buffer in H20 at pH 7.0 and at 29 o C in deoxy,oxy, and

carbonmonoxy forms: effects of ligation on the exchangeable proton resonances.

42

The data acquisition was made in the interleaf mode by switching the frequency of the irradiating field every 16 scans from the resonance of interest to an off-resonance spectral position. The time interval t 2 was 0.6 s, a value which greatly exceeds the selective longitudinal relaxation times of the exchangeable proton resonances (0.03-0.05 s). For

each N O E spectrum, 10 to 12000 transients were accumulated as differences between the irradiated and the control spectra. The proton chemical shifts are expressed as ppm relative to the water proton resonance, which i'

A. A.

Deoxy H b A

(az/3z)

Hb CO A

(azl3 z)

I / i,~',/i,l'

/i

j

/

/ t l

/

/

11.o lO.O 9.0 8.0 7.0 PPM from H20 11.0

B.

10.0

9.0 8.0 7.0 PPM from H 0

6.0

Ej

6.0

5.0

,5.0

iI

B.

I s o l a t e d a Chain

/

I s o l a t e d a Chain

/

,

11.0

,

,

100

,

/

,

9'.0 8.0 7.0 PPM from He0

6.0

/

/

tl.0 r

50 C

C.

'l/j

100

9.0 8.0 7.0 PPM from H20

6.0

5.0

I s o l a t e d /3 Chain I

I s o l a t e d /3 Chain iI

1I

/

/

/

/ 11

/ / /

! i/

/

/

J ,

r

11.0 10.0

r

~

/

J

,

,

,

9.0 8.0 7.0 6.0 5.0 PPM from He0 Fig. 2. 300 M H z 1H N M R spectra of Hb A. Isolated a and /3 chains in the deoxy form in 0.1 M phosphate buffer in H 2 0 at p H 7.0 and at 29 ° C. E represents an exchangeable proton resonance.

11.0

10.0 9.0 8.0 7.0 PPM from H20

6.0

5.0

Fig. 3 300 M H z ]H N M R spectra of Hb A and isolated a and fl chains in the carbonmonoxy form in 0.1 M phosphate buffer in H 2 0 at pH 7.0 and at 29 ° C. E represents an exchangeable proton resonance.

43 A. Control Spectrum t-LO ro

< (.D

1

I

I

I

+10.0

I

I

+8.0

i

i

+60

I

I

I

I

+4.0 +2.0 PPM from H 2 0

I

t

-2.0

I

-4.O

I

-6.0

B NOE o n t h e Resonance ot 8.2 ppm

t~

L

I

I

+t0.O

I

'

1

+8.0

/955 Tyr- C~tH , : £2H

~

I

I

+6.0

I

i

I

+4.0

e 1 2 6 Asp-/3CH 2 /9105 Leu-SzCH3

I

I

L

-2.0

+2.0

I

I

-4.0

1

I

-6.0

PPM from H20 C. NOE on the R e s o n a n c e o t 7 . 5 p p m

a105 His..../3108 Asn i

/ t

i

+10.0

elOSHis-C2H

{

I

I

+8.0

/ i_-a103 His-C4H

i

i

+6.O

t

i

t

t

]

+4.0 +2.0 PPM from H20

I

-2,O

I

t

-4.0

I

I

J

-6.0

Fig. 4. NOE difference spectra for the exchangeableproton resonancesof deoxy Hb A at + 8.2 and + 7.5 ppm in 0.] M phosphate buffer in H20 at pH 7.0 and at 29 o C. The irradiation time is 50 ms and the symbol * denotes off-resonancespillage. For resonance assignments, see the text.

44

is 4.73 p p m downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 29 ° C. The proton chemical shift scale is defined as positive in the low-field direction with respect to the water proton resonance. Results

Fig. 1 shows the exchangeable proton resonances of deoxy Hb A, H b O 2 A, and H b C O A. In deoxy Hb A, the proton resonances investigated in the present work are those from the atfl~ and cqfl 2 interfaces at +7.5, +8.2 and +9.4 p p m downfield from the water proton resonance. As previously demonstrated by this laboratory, the exchangeable proton resonance at + 9.4 p p m disappears upon the binding of ligand to Hb. Similarly, the exchangeable proton resonance at + 6.4 p p m disappears upon ligand binding to Hb. This resonance has been tentatively assigned by this laboratory to a fl-chain tertiary-structure H-bond between fl145(HC2)Tyr and f198(FG5)Val [5]. The exchangeable proton resonances at + 8.2 and + 7.5 p p m are shifted upfield by about 0.1 p p m upon ligand binding. Figs. 2 and 3 compare the exchangeable proton resonance region of the N M R spectra of H b A, isolated a a n d / 3 chains, in deoxy and CO forms, respectively. In the deoxy form (Fig. 2), none of the exchangeable proton resonances of Hb A between +6.0 and +10.0 p p m are present in isolated a or/3 subunits. The two broad resonances at approx. + 8 and + 9 p p m in these spectra are also present in deuterated solvents (results not shown) and they are the ferrous hyperfine-shifted proton resonances from the heme pockets (for a review on this subject, see Ref. 1). In the CO form (Fig. 3), the resonances of H b C O A at + 7.4 and + 8.2 p p m are also absent from the corresponding spectra of isolated a or/3 chains. The exchangeable proton resonance of H b C O A at +5.9 p p m appears to be preserved in isolated a chains, but is missing from the corresponding spectrum of isolated fl chains (Fig. 3). The NOEs observed in the present work on the inter-subunit exchangeable proton resonances of deoxy Hb A and H b C O A are summarized in Figs. 4-6. In deoxy Hb A, the irradiation of the resonance

at + 8.2 p p m results in distinct NOEs at + 3.4, + 2.8, + 2.3, - 3.5, - 4.1 and - 4.8 p p m from the water proton resonance (Fig. 4). A similar N O E pattern is observed in H b C O A upon irradiation of the resonance at +8.1 p p m (Fig. 6), but the N O E at +3.4 p p m is less intense, and those at +2.8 and - 4 . 8 p p m are shifted to +2.5 and - 5 . 0 ppm, respectively. The irradiation of the exchangeable proton resonance at + 7.5 p p m in deoxy Hb A results in specific N O E s in the aromatic proton resonance region at + 3.6, + 2.6 and + 2.3 p p m (Fig. 4). The corresponding NOEs in the aliphatic proton resonance region are less specific and a larger number of aliphatic groups appear to be affected. In the CO form, the N O E pattern for the resonance at + 7.4 p p m is similar to that in the deoxy form, but the three aromatic N O E peaks shift slightly to + 3.3, + 2.4 and + 2.0 ppm, respectively (Fig. 6). Fig. 5 summarizes the observed NOEs on the exchangeable proton resonances at +9.4 p p m which is specific to the deoxy form of Hb A. N O E s are observed in the aromatic proton reso-

A Control Spectrum

,/

'!

\ +100

+80

' + 6 0I

' + 4 0'

'+2, 0 ' PPM from H20

-2!o '-41o

_610 J

B. NOE on the Resonance at + 9 4 ppm 94 i /

t I +10.0

I

1+8!0

I

I 4-60

I

+4!0

I +2!0 i PPM from H2O

20

-40

-60

Fig. 5. N O E difference spectrum for the exchangeable proton resonance of deoxy H b A at +9.4 p p m in 0.1 M phosphate buffer in H 2 0 at pH 7.0 and 2 9 ° C . The irradiation time is 20 ms. For resonance assignments, see the text.

45

A. Control Spectrum

I-tt3 rO

qg. to

x4 I

I

I

+ 10.0

I

I

+ 8.0

I

I

+6.0

I

I

+ 4.0

I

PPM from B. N O E

onthe

Resonance

at8.1

I

+ 2.0

I

I

1

-2.0

I

-4.0

I

-6.0

H20

ppm

a126 Asp-/3CH 2/3105 Leu-82CH5

I

I

I

+10.0

I

I

+8.0

I

i

+60

I

I

+4.0

I

I

+ 2.0

L -2.0

J

l -4.0

I

I --6,0

I

PPM from H20 C. NOE on the Resonance at 7.4 ppm

a105 His .... /3108Asn

I

I

+10.0

1

I

+8.0

I

I

+6.0

a103 His-C2H /3112 Cys-peptideNH

I

I

I

+4.0

I

+2.0

I

-2.0

/3103Asn-/3CH 2

1

I

-4.0

I

I

I

-6.0

PPM from H20 Fig. 6. NOE difference spectra for the exchangeable proton resonances of HbCO A at + 8.1 and + 7.4 ppm in 0.1 M phosphate buffer in H20 at pH 7.0 and at 29 o C. The irradiation time is 50 ms and the symbol * denotes off resonance spillage. For resonance assignments, see the text.

46 nance region at +4.1, +3.5 and +2.6 ppm from the water proton resonance. The NOEs in the aliphatic proton resonance region are less specific and they affect a larger number of aliphatic groups. Discussion

The exchangeable proton resonances at +7.5 (or + 7.4), + 8.2 (or + 8.1), and + 9.4 ppm downfield from the water proton resonance have been previously proposed from this laboratory to originate from the a l f l 2 and a l f i 1 interfaces in the Hb A tetramer. This conclusion is further supported by the N M R results obtained in the present work for isolated a and /3 chains. As shown in Figs. 2 and 3, the exchangeable proton resonances at +7.5 (or +7.4), +8.2 (or +8.1) and +9.4 ppm are specific to Hb A and they are absent in the N M R spectra of isolated a and /3 subunits. Under the conditions of the N M R experiments (i.e., protein concentration 8 mM (heme)), the isolated a and /3 chains very likely exist in solution as a 2 dimers and /34 tetramers [16-18]. Hence, the absence of the exchangeable proton resonances from the corresponding 1HN M R spectra indicates that the quaternary structures of the a 2 dimers and the f14 tetramers are different from that of Hb A. These differences in the quarternary structure may contribute to the high oxygen affinity and the lack of homo- and heterotrophic allosteric effects in isolated a or/3 chains [17,19]. in the present work, we have observed specific NOEs for each of the exchangeable proton resonances investigated, in both deoxy and ligated forms (Figs. 4-6). Hence, in spite of the great effectiveness of spin diffusion in the proton longitudinal relaxation in Hb [20,21], specific NOEs can still be observed in Hb by monitoring only the initial build-up of these effects. Similar conclusions had been obtained in a preliminary study by Redfield and Huang [22] and in our laboratory for the NOEs on the heme proton resonances in deuterated solvents [23]. To gain further insight into the molecular origin of the observed NOEs, we have attempted to correlate the patterns of the NOEs to the predictions made based on the X-ray diffraction struc-

tures of deoxy Hb A and H b C 0 A [24-26] as well as based on the assignments previously proposed by this laboratory for the +9.4 and +8.2 ppm resonances [3,4]. The coordinates of the protons of amino-acid residues situated at or near the subunit interfaces (including the peptide N - H protons and the intersubunit hydrogen bonded protons) were generated as described in Experimental. E x c h a n g e a b l e proton resonance at + 8.2 p p m

The resonance at +8.2 ppm in the 1H-NMR spectrum of deoxy Hb A ( + 8.1 ppm in HbCO A) has been previously assigned in this laboratory to an H-bonded proton between a126(H9)Asp and /335(C1)Tyr at the al/31 interface. This assignment was based on the invariance of this resonance to Hb oxygenation and on its absence in Hb Philly [f135(C1)Tyr--, Phe] [4]. According to the X-ray diffraction results, the H-bonded proton a126(H9)Asp.../335(C1)Tyr is placed outside the plane of the tyrosine aromatic ring at equal distances (2.8 A) from the Col and Cc2 protons of the tyrosine residue. This local conformation predicts that the H-bonded proton of f135(C1)Tyr should have at least two NOEs in the aromatic proton resonance region of comparable intensities and linewidths. This prediction is in perfect agreement with the NOEs at +2.83 and + 2.28 ppm observed here upon the irradiation of the exchangeable proton resonance at + 8.2 ppm (Fig. 4). Moreover, these NOEs reveal that, due to the local folding of the polypeptide chain, the CE1 and Cc2 protons of/335(C1)Tyr are nonequivalent in the N M R spectra. The additional broad aromatic NOE at about + 3.4 ppm (Fig. 4) could be due to the H-bonded proton between f135(C1)Tyr and the solvent molecule anchored at the a 1/31 interface by a122(H5)His (corresponding distance of this proton to the a126(H9)Asp ... f135(C1)Tyr H-bonded proton about 3.0 A). Several aliphatic groups, such as a126(H9)AspC/3H and /3105(G7)Leu-C62 methyl protons, are situated within about 3.5 ~, from the H-bonded proton at/335(C1)Tyr and thus, these groups could contribute the NOEs observed in the aliphatic proton resonance region (Fig. 4). Hence, the analysis of the NOEs for the + 8.2 ppm exchangeable proton resonance is fully consistent with the local conformation at the

47 f135(C1)Tyr residue, and thus, the present results support the assignment of the +8.2 ppm resonance to the alfl ~ interface H-bonded proton between f135(C1)Tyr and a126(H9)Asp. Exchangeable proton resonance at + 9. 4 p p m

In a previous investigation from this laboratory, Fung and Ho [3] have found that the exchangeable proton resonance of deoxy Hb A at + 9.4 ppm was missing from the NMR spectra of deoxy Hb Kempsey (f199(G1)Asp --* Asn) and deoxy Hb Yakima (~99(G1)Asp ~ His). According to the X-ray diffraction structure of deoxy Hb A [24], the f199(G1)Asp residue is involved in the following four H-bonds: (i) the intrasubunit Hbond f199(G1)Asp...peptide NH of fll01(G3) Glu; (ii) a protein-solvent H-bond f199(G1)Asp... $3; and (iii) the t w o o~1~2 intersubunit H-bonds a 4 2 ( C 7 ) T y r . . . f 1 9 9 ( G 1 ) A s p and a97(G4)Asn...f199(G1)Asp. Hence, the replacement of the f199(G1)Asp residue in the two mutant Hbs investigated could have been expected to affect any of these four H-bonds. However, the disappearance of the + 9.4 ppm resonance upon ligand binding suggested that this resonance should originate from one of those H-bonds at the f199(G1)Asp residue which are broken in ligated Hb, namely, a42(C7)Tyr.., f199(G1)Asp or a97(G4)Asn.., f199(G1)Asp [25,27]. Fung and Ho [3] have distinguished between these two possibilities on the basis of the expected downfield shift of the tyrosine protons and thus, they have assigned the +9.4 ppm resonance to the a42(C7)Tyr ... f199(G1)Asp intersubunit H-bond. Our present NOE results support this assignment based on the following analysis. The proton in the H-bond between a42(C7)Tyr and f199(G1)Asp is found to be situated within about 4 A from the C d and Cc2 protons of a42(C7)Tyr and from the following H-bonded protons: a42(C7)Tyr . . . a97(G4)Asn; a42(C7) T y r . . . a94(G1)Asp; and a42(C1)Tyr...water solvent molecule, $2. On the other hand, no aromatic protons and only two H-bonded protons (namely, a 4 2 ( C 1 ) T y r . . , f199(G1)Asp and a42(C1)Tyor.., a97(G4)Asn) are found to be within about 4 A from the proton H-bonded between a97(G4)Asn and f199(G1)Asp. As shown in Fig. 5, the irradiation of the resonance at +9.4 ppm,

results in three distinct NOEs in the aromatic proton resonance region at + 4.1, + 3.5 and + 2.6 ppm. According to our analysis, this NOE pattern cannot result from the a97(G4)Asn...f199(G1)Asp H-bond, and thus, the most likely origin of the resonance at + 9.4 ppm is, as previously proposed, at the H-bonded proton between a42(C1)Tyr and f199(G1)Asp. Exchangeable proton resonance at + 7.5 p p m

The exchangeable proton resonance of deoxy Hb A at +7.5 ppm changes only slightly upon ligand binding to Hb A (Fig. 1). Based on this result, it has been proposed previously that this resonance should originate from the alfl 1 interface in the Hb A tetramer [3]. In the present work, we have observed NOEs for the resonance at + 7.5 ppm in the aromatic proton resonance region at + 3.6, + 2.6 and + 2.3 ppm (Fig. 4). To use these NOEs to assign the resonance at + 7.5 ppm to a specific amino-acid residue in Hb A, we have analyzed the alfl ~ intersubunit H-bonds in terms of their distances to aromatic and/or H-bonded protons. The following alfl I H-bonds were included in this analysis: M03(G10)His...fll08(H10)Asn; a31(B12) A r g . . . f l 1 2 7 ( H 5 ) G l n ; a31(B12)Arg...f1122 (GH5)Phe and a126(H9)Asp.., f135(C1)Tyr. The remaining H-bonds within the a~fl 1 interface at the f1116(G18)His and at the f130(B12)Arg have not been included in the analysis, since they are both situated close to the surface of the Hb tetramer, and thus are expected to be in fast exchange with the solvent protons. We have found that, among the a~fll H-bonds investigated, the only one whose local geometry and interproton distances match the NOE pattern observed experimentally is the H-bond a103(G10)His.../3108 (G10)Asn. The C d and Cc2 protons of al03(G10)His are situated at 2.9 ~. from the proton donated by the No2 atom of al03(G10)His to its H-bond with fll08(G10)Asn. Moreover, the amide peptide NH ~proton of fl112(G14)Cys is situated at 3.8 A from the H-bond at al03(G10)His. These three protons can thus account for the NOEs of the resonance at + 7.5 ppm and based on this fact, we can assign this resonance to the al03(G10) His... fll08(G10)Asn H-bond at the a~fll subunit interface of Hb.

48

Conclusions

References

The N O E s observed in the present work for the i n t e r - s u b u n i t low-field exchangeable p r o t o n resonances of H b A allow us to c o n f i r m previous assignments of these resonances a n d to suggest a new assignment for the resonance at + 7.5 ppm. It should be m e n t i o n e d that our previous assignm e n t s of several of these exchangeable p r o t o n resonances (i.e., + 9.4, + 8.2, + 6.4 a n d + 5.8 p p m ) were based on a c o m p a r i s o n of these resonances of H b A with appropriate m u t a n t Hbs. I n view of possible structural alterations in the H b molecule i n d u c e d by a m i n o - a c i d substitutions to sites removed from the m u t a t i o n sites in the p r o t e i n molecule, it is highly desirable to have a n i n d e p e n dent technique to c o n f i r m the spectral assignm e n t s (for a recent review on this subject, see Ref. 2). The N O E methodology discussed in this paper provides us with a n i n d e p e n d e n t c o n f i r m a t i o n of our previous spectral assignments. Moreover, we have shown that these N O E effects can be used to observe a d d i t i o n a l p r o t o n s situated at the al/~ 1 and a i r 2 s u b u n i t interfaces of H b A. These new N M R probes are currently used in our l a b o r a t o r y to o b t a i n a detailed description of i n t e r s u b u n i t interactions in n o r m a l a n d m u t a t i o n a l l y altered H b molecules in various states of ligation.

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Acknowledgements We would like to t h a n k Ms. Allison K.-L.C. Lin for her help with the H b purification, Mr. Virgil S i m p l a c a n e a u for excellent technical assistance with the N M R measurements, a n d Mr. Curtis Stehley for writing the c o m p u t e r p r o g r a m to generate p r o t o n coordinates. W e would also like to t h a n k Dr. A.G. Redfield a n d Dr. T.-H. H u a n g for sending the latter's Ph.D. thesis to us. This work is supported by a research grant from the N a t i o n a l Institutes of Health (HL-24525).