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Title: WAXS studies of the structural diversity of hemoglobin in solution.

Abstract

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCOmore » A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structure.« less

Authors:
; ; ; ; ; ; ; ; ;  [1];  [2];  [2];  [2];  [2]
  1. (Biosciences Division)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1014007
Report Number(s):
ANL/BIO/JA-70027
Journal ID: 0022-2836; TRN: US201110%%866
DOE Contract Number:
DE-AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Mol. Biol.; Journal Volume: 408; Journal Issue: 2011
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AFFINITY; CATTLE; CRYSTAL LATTICES; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; FLUCTUATIONS; HEMOGLOBIN; METHEMOGLOBIN; OXYGEN; PROBES; PROTEIN STRUCTURE; SCATTERING

Citation Formats

Makowski, L., Bardhan, J., Gore, D., Lal, J., Mandava, S., Park, S., Rodi, D. J., Ho, N. T., Ho, C., Fischetti, R. F., MCS), Northeastern Univ.), Illinois Inst. of Tech.), and Carnegie Mellon Univ.). WAXS studies of the structural diversity of hemoglobin in solution.. United States: N. p., 2011. Web. doi:10.1016/j.jmb.2011.02.062.
Makowski, L., Bardhan, J., Gore, D., Lal, J., Mandava, S., Park, S., Rodi, D. J., Ho, N. T., Ho, C., Fischetti, R. F., MCS), Northeastern Univ.), Illinois Inst. of Tech.), & Carnegie Mellon Univ.). WAXS studies of the structural diversity of hemoglobin in solution.. United States. doi:10.1016/j.jmb.2011.02.062.
Makowski, L., Bardhan, J., Gore, D., Lal, J., Mandava, S., Park, S., Rodi, D. J., Ho, N. T., Ho, C., Fischetti, R. F., MCS), Northeastern Univ.), Illinois Inst. of Tech.), and Carnegie Mellon Univ.). Sat . "WAXS studies of the structural diversity of hemoglobin in solution.". United States. doi:10.1016/j.jmb.2011.02.062.
@article{osti_1014007,
title = {WAXS studies of the structural diversity of hemoglobin in solution.},
author = {Makowski, L. and Bardhan, J. and Gore, D. and Lal, J. and Mandava, S. and Park, S. and Rodi, D. J. and Ho, N. T. and Ho, C. and Fischetti, R. F. and MCS) and Northeastern Univ.) and Illinois Inst. of Tech.) and Carnegie Mellon Univ.)},
abstractNote = {Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structure.},
doi = {10.1016/j.jmb.2011.02.062},
journal = {J. Mol. Biol.},
number = 2011,
volume = 408,
place = {United States},
year = {Sat Jan 01 00:00:00 EST 2011},
month = {Sat Jan 01 00:00:00 EST 2011}
}
  • Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps tomore » 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I 1, I 2, and I 3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme-heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I 1 intermediate is generated within 100 ps and transforms to the R-like I 2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I 3 intermediate is formed via subunit rotation, a decrease in the heme-heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I 3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme-heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.« less
  • Wide-angle x-ray scattering (WAXS) is emerging as a powerful tool for increasing the resolution of solution structure measurements of biomolecules. Compared to its better known complement, small angle x-ray scattering (SAXS), WAXS targets higher scattering angles and can enhance structural studies of molecules by accessing finer details of solution structures. Although the extension from SAXS to WAXS is easy to implement experimentally, the computational tools required to fully harness the power of WAXS are still under development. Currently, WAXS is employed to study structural changes and ligand binding in proteins; however the methods are not as fully developed for nucleicmore » acids. Here, we show how WAXS can qualitatively char- acterize nucleic acid structures as well as the small but significant structural changes driven by the addition of multivalent ions. We show the potential of WAXS to test all-atom molecular dynamics (MD) simulations and to provide insight in understanding how the trivalent ion cobalt(III) hexammine (CoHex) affects the structure of RNA and DNA helices. We find that MD simulations capture the RNA structural change that occurs due to addition of CoHex.« less