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. 2017 Mar;26(3):505-514.
doi: 10.1002/pro.3099. Epub 2017 Feb 14.

Modulation of hemoglobin dynamics by an allosteric effector

Jyotsana Lal  1 , Marco Maccarini  2 , Peter Fouquet  2 , Nancy T Ho  3 , Chien Ho  3 , Lee Makowski  1   4
Affiliations

Modulation of hemoglobin dynamics by an allosteric effector

Jyotsana Lal et al. Protein Sci. 2017 Mar.

Abstract

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide-angle X-ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.

Keywords: X-ray solution scattering; hemoglobin; neutron spin echo; protein dynamics.

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Figures

Figure 1
Figure 1
(A) The crystal structures. Shown for HbCO A (2DN3 red) human deoxy‐Hb A (4HHB blue) and R2‐state for liganded hemoglobin A (1BBB pink). Structures are aligned according to the α1β1 dimer. Figures were generated with the PyMOL program. (B) WAXS data. Shown for HbCO A, HbCO A + 3 mM IHP, deoxy Hb A and deoxy Hb A +3 mM IHP solutions taken at BioCAT at the APS. Deoxy Hb A WAXS data is slightly offset in y for a clearer display. The concentration of the proteins is 10 mg/mL. The intensity scale is arbitrary. (C) P(r) calculated from WAXS data. The calculation of p(r) in (C) are based on the measured WAXS data in (B).
Figure 2
Figure 2
Polarization analysis. Shown for HbCO A (IHP) in solution in deuterated water (D2O). The fractions of coherent and incoherent scattering are retrieved from the polarization analysis as a function of the scattering vector q. Measured with the NSE spectrometer IN11 (Institut Laue‐Langevin). (a) Top panel displays the polarization analysis before subtraction of the D2O buffer. (b) Bottom panel displays the polarization analysis after subtraction of the D2O buffer.
Figure 3
Figure 3
Coherent domain. Normalized structural correlation functions S coh(q,t)/S(q,0) = Sij(q,t)/S(q,0) of HbCO A (IHP) in D2O, as measured with the NSE spectrometer IN11 (Institut Laue‐Langevin) at five momentum transfers q at a protein concentration of 100 mg/mL and a temperature of 288 K. Continuous lines indicate fits of the data to a single‐exponential decay function.
Figure 4
Figure 4
Incoherent domain. Normalized self correlation functions S inc(q,t)/S(q,0) = Sii(q,t)/S(q,0) of HbCO A (IHP) in D2O, as measured with the NSE spectrometer IN11 (Institut Laue‐Langevin) at four momentum transfers q at a protein concentration of 100 mg/mL and a temperature of 288 K.
Figure 5
Figure 5
Relaxation times. Shown for both the coherent and incoherent domains. In the coherent scattering regime (q < 0.3 Å−1), the relaxation time corresponds to that in Eq. (2), based on a single exponential fit (inset shows D eff vs. q near 0.2 Å−1) versus momentum transfer q. In the incoherent scattering regime (q > 0.3 Å−1), the relaxation time corresponds to τ KWW as defined in the discussion following Eq. (4) (Table 1).
Figure 6
Figure 6
Root mean square displacement (RMSD) of hydrogens. RMSD of HbCO A and HbCO A + IHP solutions shown as a function of correlation time for the incoherent neutron scattering. For times less than about 1 ns, the RMSD is lower in the presence of IHP; for correlation times greater than a nanosecond, IHP induces an increase in RMSD.
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