Subunit Disassembly and Unfolding Kinetics of Hemoglobin Studied by Time-Resolved Electrospray Mass Spectrometry

Self Cite

101

The native structure of hemoglobin (Hb) comprises two ␣-and two ␤-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded ␣-and ␤-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric ␣-and ␤-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded ␣-and ␤-globin. However, some unexpected features are observed, e.g., a heme-deficient ␤-globin dimer that represents an off-pathway intermediate. Monomeric ␤-globin is capable of binding heme before forming a complex with an ␣-subunit. This observation suggests that holo-␣-apo-␤ globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes. (J Am Soc Mass Spectrom 2007, 18, 8 -16)

Section: Hemoglobin Refolding Monitored By Esi-mssupporting

confidence: 83%

“…These dimeric structures become unstable below pH 4, forming highly unfolded monomeric apo-subunits around pH 3. This stepwise process resembles the denaturation mechanism observed in kinetic ESI-MS experiments, where unfolding°was°triggered°by°a°sudden°pH°jump° [61].…”

Section: Reversible and Irreversible Denaturation Of Hemoglobinsupporting

confidence: 58%

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

Boys

Konermann

2007

Self Cite

101

“…Thus the levels of dimers and monomers in solution are expected to be very low. Nevertheless, all ESI MS studies of Hb show substantial levels of dimers and monomers in the spectrum [32,35,[42][43][44].…”

Section: Mass Spectra Of Hemoglobinmentioning

confidence: 99%

“…Each of the subunits can be in the hologlobin (heme-bound) form (␣ h ␣ ␣ and ␤ h ␤ ␤ ) or apoglobin form (␣ o ␣ and ␤ o ␤ ␤ ) [30]. ESI can form multiply protonated gas-phase ions of these noncovalently bound hemoglobin complexes, provided that the solvent composition and instrument conditions are appropriately chosen [31][32][33][34]. Hemoglobin provides a model case where the properties of the gas-phase dimer subunits and tetramers can be compared directly with their monomeric subunits.…”

mentioning

confidence: 99%

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

Wright

Douglas

2009

The conformations of gas-phase ions of hemoglobin, and its dimer and monomer subunits have been studied with H/D exchange and cross section measurements. During the H/D exchange measurements, tetramers undergo slow dissociation to dimers, and dimers to monomers, but this did not prevent drawing conclusions about the relative exchange levels of monomers, dimers, and tetramers. Assembly of the monomers into tetramers, hexamers, and octamers causes the monomers to exchange a greater fraction of their hydrogens. Dimer ions, however, exchange a lower fraction of their hydrogens than monomers or tetramers. Solvation of tetramers affects the exchange kinetics. Solvation molecules do not appear to exchange, and solvation lowers the overall exchange level of the tetramers. Cross section measurements show that monomer ions in low charge states, and tetramer ions have compact structures, comparable in size to the native conformations in solution. Dimers have remarkably compact structures, considerably smaller than the native conformation in solution and smaller than might be expected from the monomer or tetramer cross sections. This is consistent with the relatively low level of exchange of the dimers. . Electrospray ionization has allowed the study of protein ions in the gas phase, free of water. In the absence of water, monomeric proteins can adopt compact conformations similar to the solution conformations, but also extended conformations far removed from the native state [2,3]. Much of the current understanding of the "size" of protein ions in the gas phase comes from physical methods of measuring collision cross sections using ion mobility spectrometry at pressures of a few Torr [4 -7], at atmospheric pressure [8], or at pressures of ca. 1 ϫ 10 Ϫ3 Torr [7,9,10]. Measurements of ion kinetic energy loss in triple quadrupole systems have also been used to determine collision cross sections of proteins [11][12][13] and protein-ligand complexes [11,14,15]. Chemical probes of protein conformation have been used as well, with much of the work using gas-phase hydrogen deuterium exchange (H/Dx) of trapped ions, either in linear [16 -19] or 3D [20] quadrupole ion traps, or ion cyclotron resonance (ICR) mass spectrometers [21][22][23][24][25].The study of the structure of monomeric proteins in the gas phase has been extended to assemblies containing more than one protein. Ion mobility measurements of complex macromolecular protein complexes at atmospheric pressure [8] or at a pressures of ca. 1 ϫ 10 Ϫ3 Torr have shown some success [9,10]. The resolution of these experiments, ca. 5-20, is lower than more conventional mobility measurements at pressures of a few Torr.Protein-protein and protein-ligand complexes can be studied by gas-phase H/Dx of trapped ions. The pressures of deuterating reagent in linear trap experiments can be ϳ5 ϫ 10 Ϫ3 Torr, ca. 10 3 times greater than used in 3D traps [20], 10 1 to 10 2 times greater than used in mobility experiments [26,27], and 10 3 to 10 4 times greater than in ICR experiments [21][22][2...

“…The complementary advantages of microfluidic liquid handling and ESI-MS based analysis make these devices a natural answer to the challenges of proteomic studies. One 'underrepresented' application is rapid microfluidic mixing for time-resolved studies by ESI-MS. Time-resolved ESI-MS (TR ESI-MS) is a powerful approach for the characterization of shortlived, 'transient' intermediates in biochemical reactions [3][4][5][6][7][8][9][10], which are critical to the biological activity of proteins, from enzymatic reactions [7,11] to protein folding [12,13].…”

mentioning

confidence: 99%

A versatile microfluidic chip for millisecond time-scale kinetic studies by electrospray mass spectrometry

Rob

Wilson

2009

Self Cite

An electrospray coupled microfluidic reactor for the measurement of millisecond time-scale, solution phase kinetics is introduced. The device incorporates a simple two-channel design that is etched into polymethyl methacrylate (PMMA) by laser ablation. The outlet of the device is laser cut to a sharp tip, facilitating low dead volume 'on chip' electrospray. Fabrication is fast, straightforward and highly reproducible, supporting rapid prototyping and large-scale reproduction. Device performance is characterized using a cytochrome c unfolding reaction. Unfolding processes with rates in excess of 30 s Ϫ1 are easily measured, including the appearance of a 'native-like' intermediate that is maximally populated 180 ms post reaction initiation. To extract reliable rates from the data, a theoretical framework for the analysis of kinetics acquired under square-channel laminar flow is introduced. (J Am Soc Mass Spectrom 2009, 20, 124 -130)