Downloaded via YORK UNIV on December 11, 2018 at 06:09:04 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Chapter 12
An Approach to NMR Treatment of Structural Perturbations in Paramagnetic Proteins too Big for Solution Structure Determination 1
2
1
Judith M.Nocek ,KaiHuang ,and Brian M. Hoffman 1
2
Departments ofChemistryandStructuralBiology, NMR Facility, Northwestern University, 2145 North Sheridan Road, Evanston, IL 60208
The classic description of the quaternary structure change accompanying cooperative ligand binding by hemoglobin (Hb) assumes the existence of two stable tetrameric, quaternary structural classes: the fully-oxygenated (R-state) and the completely deoxygenated (T-state) structures. To characterize the structures that actually occur in solution, to improve the ability of the NMR experiment to distinguish members within a structural class, and ultimately to use this improved ability to assign the structures of intermediate ligation states to members within such families, we have been developing a new approach in which experimental pseudocontact shifts are analyzed to obtain a site-specific description of structural changes produced by mutations in the intradimer interface.
© 2003 American Chemical Society
Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
227
228 Historically, the structure changes accompanying cooperative ligand binding by the hemoglobin tetramer (Hb) have been taken to involve two stable quaternary structures: the R-structure assumed by fully oxygenated Hb and the Tstructure assumed by completely deoxygenated protein. Although details of the mechanism by which these structures interconvert is not yet fully understood, Τ R conversion involves a change in the orientation of the two heterodimers (α, β, and α β ) of Hb with respect to each other. Characterization of the intermediate ligation states is a critical component of a full understanding of the mechanism whereby the quaternary structure of Hb changes upon uptake and release of oxygen. Although these intermediates are present only in small amounts in the erythrocyte, making them difficult to prepare and characterize, studies with them(/-5) indicate that the mechanism of oxygen uptake/release is far more complicated than the simple analysis implies. Recent crystallographic studies have shown that more than two quaternary structures (designated R and R ) are, in fact, energetically accessible to the ligatedstate of tetrameric human hemoglobin«5) while examination of the xray structures for a set of mammalian Hbs has shown that the ligated state can actually adopt any of an ensemble of structures that encompasses both the Rstructure and the R -structure.(6) Together, these results have prodded a re examination of the structural relationships and the thermodynamic linkages that lead to cooperative ligand binding.(7) To characterize the structures that actually occur in solution, to improve the ability of the NMR experiment to distinguish members within a structural class, and ultimately to use this improved ability to assign the structures of intermediate ligation states to members within such families, measurements that yield geometric information are needed with site-specific resolution. While significant progress has been made toward obtaining a complete NMR structure of Wo,(8-14) the four probes assigned to exchangeable protons on aromatic residues in the α,β and α,β, interfaces are still the primary NMR signatures for assessing the quaternary state of the protein in solution.(//) Clearly, more extensive assignments are needed. In a first effort to identify new probes, we recently incorporated N into the β-chain Val residues of R-structure carboxy-Hb^VlM) and fluorometHb$VlM), and used a novel assignment strategy, based on comparison of predicted and measured pseudocontact shifts, to identify the amide backbone signals in N-val labeled Hb.(/4) The 17 valine residues in the β-chain are distributed throughout the chain and several are close enough to the heme that they exhibit large pseudocontact shift increments. We,(7< 75) like others,(/2,13,16-24) have found that such experimental pseudocontact chemical shift increments can be utilized to obtain site-specific structural information for paramagnetic metalloproteins. In particular, we have shown that these probes in Hb are sensitive to the quaternary structure changes induced by the addition of allosteric effectors.(/4) We now discuss the use of these resonances in characterizing the structure of the Hb$W37E) interface mutant, whose functional(25,26) and structural properties(27) indicate that it exemplifies a new class of high-affinity, unligated tetramers, denoted T . We find that this mutation causes widespread changes in the β chain, supporting the idea that the structure of the mutant is different from that of native R-structure carboxy-Hb. As the chemical shift differences (Δδ}) cannot at present be used to derive quantitative information about the changes in atomic 2
2
2
2
2
,5
15
hj
Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
229
position of the nuclei, we are developing a protocol for deriving a site-specific geometric description of the mutation-induced structural changes from the pseudocontact shift increments (Δδ, ). In this progress report, we present results that support the view that the liganded state of the Hb(PW37E) mutant adopts a structure that indeed differs from the native R-structure. ρ
General Approach Imagine modifications, made either by chemical means or by mutagenesis, to a large paramagnetic protein of known structure. If that protein is too large to obtain a complete NMR structure, can one nevertheless make use of a limited set of NMR assignments to assess the effects of the modification on that structure? Here we describe an approach in which the presence of a paramagnetic center bound to a protein is exploited firstly to compare the overall quaternary state of the modified protein with the native protein, and secondly to quantify the structural perturbations resulting from the modification. This requires that a suitable set of NMR probes have been assigned, and that the chemical shifts of these probes can be measured in both the paramagnetic state and a suitable diamagnetic state having a similar structure.
Pseudocontact Shifts bs
The total observed chemical shift (ô° ) for a paramagnetic system can be partitioned into diamagnetic (ôj ) and paramagnetic contributions, the latter being separable into pseudocontact (δ ) and Fermi contact (ô, ) components.(Equation d,a
ρ
FC
1)
+6/ +
V * = 6^
(1)
For protons that are not covalently-linked to the paramagnetic center, the Fermi contact term is negligible (ô, = 0), leaving only the pseudocontact contribution to the paramagnetic shift. In this case, experimental pseudocontact shift increments for any given atom are calculated by subtracting the chemical shift for a suitable diamagnetic reference sample from those of the paramagnetic sample (ô, = ô, FC
p
obs
The pseudocontact shifts for the ι-th proton can also be calculated (17,23) from a known x-ray structure through use of Equation 2.
&XAX
( 3 c o r t l ) + Axjtg (CQS26, ήαφ) r
3NR?
Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
(2)
230
Here Δ χ and Δ χ are the axial and rhombic contributions to the magnetic susceptibility and Ν is Avogadro's number. The distance (R;) between the paramagnetic center and the /-th proton, and the polar and azimuthal angles (Θ, and φ,) describe the location of the /-th proton within the framework of the susceptibility tensor.(Figure 1) In its most general form, the pseudocontact shift for the z-th proton ( δ ) includes contributions from all the paramagnetic centers in a protein (Equation 3), ΑΧ
ΛΗ
p
with the pseudocontact shift for each magnetic center being determined by Equation 2. In the case of Hb, there are four magnetic centers, each within a different subunit. As large changes in the quaternary state of Hb are linked to changes in the tertiary structure of a given subunit, the contribution to the pseudocontact shift from the heme within the subunit where the probe resides usually provides a good approximation of the total shift. The exception to this would be probes that are situated between two (or more) of the paramagnetic centers (e.g., Val 98 that is near the afi intrasubunit interface). Structural perturbations which cause a nucleus to move relative to the susceptibility tensor frame give rise to an increment in the pseudocontact shift (Δδ ). The sensitivity of the pseudocontact shift to such a structural modification varies with the location of the probe nucleus. Figure 2 illustrates the well-known angular dependence for the calculated pseudocontact shift increment ( δ , Equation 2) for a probe nucleus on the surface of a sphere at a distance of 10 A from a paramagnetic center with an axial g-tensor (Δχ = 1.0 χ 10" nrVmol; Δ χ = 0 mVmol). The magnitude of the shift varies periodically with the angular (Θ) position of the probe on the surface of the sphere. The maximum upfield shift occurs at θ = π/2, while the maximum downfield shift occurs at θ = 0, π. With a rhombic susceptibility tensor, the pseudocontact shift varies with both the polar (Θ) and azimuthal (φ) angles. Figure 3A presents a set of contour plots describing the angular dependencies for δ for a probe nucleus at 10 A from a paramagnetic center whose tensor properties are chosen to be comparable to those reported for cyanomet-Hb ( Δ χ = 1.5 χ 10" mVmol; άχ = -0.5 χ ΙΟ" m /mol). For all values of φ, the maximum upfield shift occurs at θ = π/2 and the maximum downfield shift occurs at θ = 0, π, as is observed for the axial limit (φ = 0). The pseudocontact shift shows a strong dependence on the azimuthal angle only when θ is near π/2. 2
p
ρ
9
ΑΧ
ΚΗ
ρ
9
ΑΧ
9
3
ΚΗ
Site-Sensitivity Factors If the mutation-induced structural change in an R ., vector is small in comparison to the R . vector, the mutation-induced increment in the pseudocontact shift of the z-th atom, Δ δ = [ô, (mutant) - ô^wild type)], can be written as the M
M f
ρ
p
Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Telser; Paramagnetic Resonance of Metallobiomolecules ACS Symposium Series; American Chemical Society: Washington, DC, 2003. 0
Figure 1: A. Coordinate system describing the magnetic axes (x \ y \ ζ ') and the iron-centered pseudoaxes system derived from the x-ray coordinates of oxyHb. The two coordinate systems are related by the Euler transformation angles (α, β, and γ). Β. Components describing ARf), the mutation-induced structural change in the R vector.
232
Figure 2: Dependence of the predicted pseudocontact shift and sensitivity factors on din the axial limit. Parameters: Δχ = 1.0χ Iff m /mol; R = 10 À 9
3
ΑΧ
Aôf = Sgdr + S drx + S dr s
e
9
(4)
m
weighted sum of terms arisingfrommotions of the /'-th atom along three mutuallyorthogonal directions,(24) as shown in Figure IB: (i) along the R . vector (dR,); (ii) perpendicular to this vector and within the plane defined by χ and R . (dR „); (iii) perpendicular to this plane (dR^). We refer to the coefficients (S , S , and S
