Anal. Chem. 2001, 73, 4763-4773
Detection of Multiple Protein Conformational Ensembles in Solution via Deconvolution of Charge-State Distributions in ESI MS Andras Dobo and Igor A. Kaltashov*
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
Monitoring the changes in charge-state distributions of protein ions in electrospray ionization (ESI) mass spectra has become one of the commonly accepted tools to detect large-scale conformational changes of proteins in solution. However, these experiments produce only qualitative, lowresolution information. Our goal is to develop a procedure that would produce quantitative data on protein conformational isomers coexisting in solution at equilibrium. To that end, we have examined the evolution of positive ion charge-state distributions in the ESI spectra of two model proteins, r-helical myoglobin (Mb) and β-sheet cellular retinoic acid binding protein I (CRABP I), as a function of solution pH. A detailed analysis of the charge-state distributions over a wide range of pH (2.6-8.5) suggests that each spectrum (i.e., relative ion abundance I vs its charge state n) can be approximated as a linear combination of a limited number of basis functions Bi(n), i.e. I(n) ) ∑biBi(n). These basis functions (approximated as normal, or Gaussian, distributions) are not significantly affected by the pH variations; however, their relative intensities (coefficients bi) exhibit strong pH dependence giving rise to complicated overall charge-state distributions. Analysis of the experimental data, aided by the vast existing body of knowledge of Mb and CRABP I conformational properties (both structure and dynamics) leads to a conclusion that each basis function in fact represents a single conformational isomer. Average charge state corresponding to each basis function (e.g., position of the maximum of Bi(n) on the protein ion charge scale n) characterizes the conformer‘s overall shape (most likely, projected surface area). The width of each basis function (i.e., standard deviation of the normal distribution) represents the conformer’s heterogeneity. Overall, this technique is suitable for analysis of complex mixtures of protein conformational isomers in solution and complements existing experimental methods that are used to study macromolecular dynamics by characterizing protein shape in solution (e.g., scattering techniques).
environmental changes remains the focus of extensive research efforts, as conformation and dynamics are the key features that determine protein function.1 While NMR spectroscopy remains the only technique capable of providing structural information at atomic resolution, as well as probing dynamics on multiple time scales (from subnanoseconds to days),2 it has certain disadvantages. These include molecular weight limitations, problems with analyzing complexes containing paramagnetic ligands, difficulties with correlating site-specific dynamics with distinct conformers, etc. Most other techniques that are often employed to study protein conformational dynamics usually monitor either cumulative changes of the secondary structure (CD in the far-UV region,3 FT-IR and Raman spectroscopy4) or specific markers of dynamic events (e.g., fluorescence measurements to assess exposure of aromatic residues to solvent5). Another experimental approach that is now gaining wide acceptance as a means to characterize protein dynamics by probing its overall shape is small-angle X-ray scattering (SAXS), as well as closely related small-angle neutron scattering (SANS)6 and dynamic light scattering (DLS). Although the solution scattering has been viewed traditionally as a method to provide gross structural information (e.g., radius of gyration, interatomic distance distribution, etc.), recent advances in theory have led to introduction of several methods of extracting lowresolution macromolecular structures from the scattering profiles.7,8 Perhaps, one of the greatest challenges in protein characterization by SAXS is detection and characterization of individual conformational states in a polydisperse system. Although this challenge can be addressed in some cases by applying a singular value decomposition (SVD) algorithm to scattering profiles,9,10 this analysis is greatly facilitated if supplementary structural/thermodynamic data are available.11 (1) (2) (3) (4) (5) (6) (7) (8)
Design of experimental methods aimed at probing protein three-dimensional structure and its evolution as a result of * Corresponding author: (tel) (413) 545-1460; (fax) (413) 54504490; (e-mail)
[email protected]. 10.1021/ac010713f CCC: $20.00 Published on Web 09/15/2001
© 2001 American Chemical Society
(9) (10) (11)
Price, N. C. Biotechnol. Appl. Biochem. 2000, 31, 29-40. Ishima, R.; Torchia, D. A. Nat. Struct. Biol. 2000, 7, 740-743. Pelton, J. T.; McLean, L. R. Anal. Biochem. 2000, 277, 167-176. Barron, L. D.; Hecht, L.; Blanch, E. W.; Bell, A. F. Prog. Biophys. Mol. Biol. 2000, 73, 1-49. Selvin, P. R. Nat. Struct. Biol. 2000, 7, 730-734. Perkins, S. J.; Ashton, A. W.; Boehm, M. K.; Chamberlain, D. Int. J. Biol. Macromol. 1998, 22, 1-16. Svergun, D. I. Biophys. J. 1999, 76, 2879-2886. Chacon, P.; Diaz, J. F.; Moran, F.; Andreu, J. M. J. Mol. Biol. 2000, 299, 1289-1302. Chen, L.; Hodgson, K. O.; Doniach, S. J. Mol. Biol. 1996, 261, 658-671. Segel, D. J.; Bachmann, A.; Hofrichter, J.; Hodgson, K. O.; Doniach, S.; Kiefhaber, T. J. Mol. Biol. 1999, 288, 489-499. Segel, D. J.; Eliezer, D.; Uversky, V.; Fink, A. L.; Hodgson, K. O.; Doniach, S. Biochemistry 1999, 38, 15352-15359.
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Mass spectrometry is now becoming an increasingly popular technique that is often used to probe protein higher order structure and dynamics under a variety of conditions. Following the pioneering work by Chowdhury et al.12and Loo et al.,13 electrospray mass spectrometry (ESI MS) has been used to probe qualitatively protein conformational changes as a function of solvent composition. Consistent with Fenn’s model of protein ion desorption during the electrospray process,14 less structured proteins in solution usually produce higher charge-state ions, as increased protein surface area allows for accommodation of a relatively large number of charges. In contrast, proteins that retain more or less compact structure in solution cannot accommodate as many charges upon desorption to the gas phase because of the limitations imposed by electrostatic repulsion. As a result, these ions have low charge state and the corresponding peaks appear in the high-m/z region in the spectrum. Although Fenn’s model provides adequate qualitative description of these processes, the numerical predictions are difficult make.15 Validity of the qualitative description of the protein ion desorption has been supported by numerous observations of dramatic shifts in chargestate distribution as a result of changing the protein environment. Our own recent measurements of the amide hydrogen/deuterium (H/D) exchange of proteins under mildly denaturing conditions also supports the notion that the low- and high-charge-state protein ions represent different conformers in solutions.16 Thus, the H/D exchange kinetics measured using these two groups of ions is markedly different, suggesting that more protected (structured) proteins give rise to the low-charge-state (high-m/z) ions. Obviously, this behavior is observed only if the protein refolding rate is significantly lower than the intrinsic exchange rate (i.e., in the case of “slow” equilibrium). However, the measurements based solely on monitoring protein ion charge-state distributions are traditionally considered to produce only qualitative data at low resolution. Therefore, the amide H/D exchange kinetics measurements17,18 are almost always favored when multiple (>2) conformers are present in solution. In the present work, we introduce and evaluate a method of detecting and characterizing multiple conformational isomers of proteins by analyzing charge-state distributions of protein ions in ESI mass spectra. Our results indicate that the size and heterogeneity of each conformational state can be characterized in situations when several such states are populated. Analysis of the ESI MS data for the two model proteins used in this initial study produces a picture of dynamic events that is fully consistent with the existing knowledge of their behavior (resulting from the extensive research efforts during the past decade). (12) Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 9012-9013. (13) Loo, J. A.; Loo, R. R.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101-105. (14) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-535. (15) Kebarle, P.; Ho, Y. In Electrospray ionization mass spectrometry: fundamentals, instrumentation and applications.; Cole, R. B., Ed.; Wiley: New York, 1997; pp 3-63. (16) Eyles, S. J.; Dresch, T.; Gierasch, L. M.; Kaltashov, I. A. J. Mass Spectrom. 1999, 34, 1289-1295. (17) Miranker, A.; Robinson, C. V.; Radford, S. E.; Dobson, C. M. FASEB J. 1996, 10, 93-101. (18) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135-146.
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EXPERIMENTAL SECTION Materials. Apo- and holomyoglobin (horse skeleton muscle) were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. A sample of β-sheet cellular retinoic acid binding protein I (CRABP I) was generously provided by Prof. Lila M. Gierasch (University of Massachusetts). All solvents and buffers used in this work were of analytical grade or higher. Mass Spectrometry. All mass spectra were acquired on a JMS-700 MStation (JEOL, Tokyo, Japan) two-sector mass spectrometer equipped with a standard ESI source. The ESI MS samples were prepared by diluting protein stock solution (200 µM in 10 mM NH4CH3CO2) in 5 mM NH4CH3CO2 buffer solution whose pH was adjusted to a desired level with NH4OH or CH3CO2H. Final protein concentration was 2 µM. All solutions were kept at room temperature (24 °C) prior to analyses. The samples were introduced into the ESI source at flow rates of 5 µL/min. All ESI source parameters and settings (temperature in the desolvation region, electrostatic potentials on all ion optics elements, and settings of the quadrupole ion guide) were kept constant throughout all measurements to avoid any possible changes in ion desorption and transmission conditions. All spectra were acquired by scanning the magnet at a 5 s/decade rate. To ensure high signal-to-noise ratio, 100-200 scans were typically averaged to record each spectrum. Data Processing. ESI spectra were processed (curve fitting) using Microcal Origin (Microcal Software, Inc., Northampton, MA) software. The ion peak intensity cutoff level was set at 3% on the relative abundance scale. The initial parameters of the basis functions, i.e., population central (νi0), population standard deviation (σi), and coefficients bi were introduced manually, followed by iterative optimization using the built-in Simplex method to obtain minimal χ2 value. Circular Dichroism. All circular dichroism (CD) spectra were acquired with a J-715 (Jasco, Tokyo, Japan) spectropolarimeter. RESULTS AND DISCUSSION Scope of the Present Work. Partial unfolding or emergence of non-native conformations of proteins in solutions can be induced by a variety of factors, including variations in solvent composition (e.g., addition of chaotropes, such as detergents, alcohols, and urea). Among the plethora of factors that can modulate protein structure and its stability, solution pH is perhaps one of the most important. Indeed, the pH adjustment within certain cellular compartments is a universal mechanism that often triggers complicated protein interaction cascades by inducing specific conformational changes. One of the examples of such pH-driven processes is a receptor-mediated endocytosis,19 in which acidification by vacuolar (H)-ATPases is essential for ligand-receptor dissociation and receptor recycling. Therefore, the scope of this work has been limited to studies of acid-induced conformational isomers. Two proteins were chosen as model systems for this initial study. Myoglobin (Mb) is a single-chain globular protein consisting of eight R-helices (total helical content of the native conformation is 75%20) and a noncovalently linked heme group. Acid unfolding of both apo- and holoforms of Mb (aMb and hMb) has been characterized extensively in the past using a variety of (19) Clague, M. J. Biochem. J. 1998, 336, 271-282. (20) Evans, S. V.; Brayer, G. D. J. Mol. Biol. 1990, 213, 885-897.
Figure 1. Positive ion ESI mass spectra of hMb (A-E) and aMb (F-J) acquired at pH 7.4 (A, F), 4.5 (B, G), 4.0 (C, H), 3.5 (D, I), and 2.5 (E, J). Intact hMb ion peaks are indicated with filled squares.
experimental techniques. Most studies have focused on aMb due to experimental problems associated with the heme prosthetic group. In the present study, we use both aMb and hMb, since the heme group stabilizes the protein structure and also serves as a reporter of the protein structural integrity. Another model protein used in our work is CRABP I. This protein is unique in that it is almost an entirely β-sheet protein (most other proteins whose structures have been solved are helical, like Mb or R/β). Acid-induced unfolding of this protein has also been studied in the past by several groups using CD, NMR,21 and mass spectrometry.16 Acid-Induced Unfolding of Myoglobin. ESI mass spectra (positive ion mode) of both hMb and aMb were obtained over a pH range 2.5-8.6 (Figure 1). The hMb spectra exhibit a very (21) Liu, Z. P.; Rizo, J.; Gierasch, L. M. Biochemistry 1994, 33, 134-142.
narrow charge-state distribution at pH 4.5 and above (in fact, each spectrum contains only two ion peaks, charge states +8 and +9). The source conditions were selected in these experiments (and used in all subsequent experiments) such that no gas-phase protein-heme dissociation is observed. Further decrease of solution pH (i.e., to pH 4) results in (i) large-scale conformational changes, as manifested by the appearance of the highly protonated (i.e., low-m/z) protein ions and (ii) partial dissociation of the heme group from the protein. The intensity ratio of holo- versus apoprotein ion peaks depends greatly on the ion charge state. Thus, this ratio exceeds 1.3 for the charge states +8 and +9, but drops off sharply to less than 0.2 at higher charge states, consistent with the notion that the heme group can be used as a reporter of the integrity of Mb higher order structure. Further decrease of solution pH leads to (i) continuous increase of the Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
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average charge state of protein ions and (ii) disappearance of the protein-heme complex ions. Unlike hMb, the aMb spectrum exhibits multimodal character even at neutral pH (Figure 1F). In addition to +9 and +8 ions, a wide distribution of less abundant ion peaks is seen in the spectrum at charge states ranging from +10 to +23. This is indicative of the presence of multiple conformational isomers of aMb coexisting in solution under equilibrium and is consistent with earlier observations by Wang22 and Konermann.23 Acidification of the aMb solution results in diminished abundance of the low-charge-state ions (+8 and +9) and continuous increase of the abundance of the high-charge-state ions. The most abundant ion peak in the aMb spectrum at pH 2.5 corresponds to a +20 charge state, while the charge-state envelope extends to +28. At this pH, the aMb spectrum is practically indistinguishable from that of the hMb, fully consistent with the expectation that any interaction between the heme group and the acid-destabilized form of the protein would be minimal. Spectra of both hMb and aMb clearly exhibit tendencies that are consistent with Fenn’s model of electrospray ionization.14 Thus, tightly folded protein molecules are expected to have significantly smaller projected areas (as compared to less structured protein molecules), resulting in accommodation of a fewer number of charges on the protein surface at the ion evaporation stage. However, most globular proteins are known to have multiple folding intermediate states (not just folded and unfolded states), some of which may have only minor structural differences. Subtle conformational changes are unlikely to lead to significant variations in the overall shape of the protein (e.g., projected areas, and, therefore, in the average number of charges accommodated upon protein ion desorption). As a result, the charge-state distributions corresponding to various protein conformational isomers may be unresolved or poorly resolved (i.e., two or more different conformers may give rise to ions carrying the same number of charges). Thus, close examination of the protein ion charge-state distributions in Figure 1 reveals in many cases the presence of multiple (>2) maximums on continuous distributions (e.g., +9, +11, and +15 for aMb at pH 4.0; see Figure 1H). This indicates the presence of at least three different protein conformers populated with significant Boltzmann weight. However, neither the actual number of protein conformational isomers nor their characteristics can be determined using this straightforward approach. To circumvent this problem, we introduce and evaluate a new approach to analyzing complex ESI spectra of proteins. Presented below is a brief outline and a rationale of our approach. Ionization of Protein Conformational Isomers and Their Contributions to the ESI Spectrum. Our interpretation of the positive ion ESI spectra of aMb and hMb is based on Fenn’s semiempirical model of the ESI process.14 We assume that that, under certain conditions, each conformational state of the protein would produce a specific charge-state distribution. These specific distributions will be referred to as “basis functions” Bi(n) (where n is a charge state and “i” refers to a particular protein conformer). The ESI spectrum obtained under these conditions would then be a sum of contributions from each conformer present in solution, i.e., a sum of weighted basis functions: (22) Wang, F.; Tang, X. Biochemistry 1996, 35, 4069-4078. (23) Konermann, L.; Douglas, D. J. Rapid Commun. Mass Spectrom. 1998, 12, 435-442.
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I(n) )
∑b B (n) i
i
where “weighting” coefficients bi reflect both concentration and ionization efficiency of conformer i. Therefore, for the purposes of our analysis, we can treat each conformational isomer as a distinct species, while keeping in mind that all conformers have the same molecular weight and exist under equilibrium with each other. Although the overall protein shape (projected area in Fenn’s terminology) is a very important determinant of the corresponding ion’s average charge state, it certainly is not the only one. Variation in solvent composition may affect ion charge state by (i) altering the desorption process (e.g., alteration of the surface tension of sprayed droplets and solvent dielectric constant)24 or (ii) inducing charge-transfer ion molecular reactions that involve protein ions and molecules of evaporated solvent in the ESI interface region.25 Alterations of the solvent composition in the present work have been achieved by adding low-millimolar or submillimolar amounts of either CH3CO2H or NH4OH to a buffer stock solution (5 mM NH4CH3CO2 in H2O). Therefore, it is very unlikely that either the surface tension of the sprayed droplets or the ion chemistry in the interface region would be affected by pH variations alone. Likewise, the solvent dielectric constant is not expected to vary significantly as a function of pH, since the ionic strength of the solution does not exhibit significant changes. Historically, pH changes had also been thought to induce variations in observed charge-state distributions by altering the ionization state of the proteins in the bulk solution.26 However, multiple ESI MS “titration” studies conducted in the past decade had lead to an unequivocal conclusion that it is the ion desorption process that dictates the ionization state(s) of macromolecules detected with ESI MS.14,27,28 These arguments are supported by our own observations of the behavior of hMb in the pH range 4.5-8.6. We know that hMb is stable in this pH range (under the conditions used for ESI MS measurements) both from our own near-UV CD measurements (i.e., by monitoring the Soret band) and from the published studies on the stability of this protein in “medium” ionic strength solutions.29 Not surprisingly, the ESI spectra of hMb exhibit minimal variations within this pH range (Figure 1A and B). On the basis of these arguments, we now assume that the +9 and +8 ions peaks in the hMb spectra in the pH range 4.5-8.6 represent one basis function. We chose to represent this function mathematically as a normal distribution, i.e.
Bhi (n) )
1
0 2
2
x2πσi
e(-(n-νi ) /2σi )
(1)
2
where the subscript is an identifier of the basis function in the (24) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287-2294. (25) Schnier, P. D.; Gross, D. S.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1995, 6, 1086-1097. (26) Guevremont, R.; Siu, K. W. M.; Le Blanc, J. C. Y.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 216-224. (27) Kelly, M. A.; Vestling, M. M.; Fenselau, C. C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143-1147. (28) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120-1130. (29) Chi, Z.; Asher, S. A. Biochemistry 1998, 37, 2865-2872.
Figure 2. Curve fitting of positive ion charge-state distributions in ESI mass spectra of hMb (A-C) and aMb (D-F) acquired at pH 7.4 (A, D), 4.0 (B, E), and 2.5 (C, F). Experimental data points are shown with squares (9 for intact hMb ions and 0 for aMb ions). Gaussian curves represent the weighted basis functions used for curve fitting (shaded for hMb). Thick solid lines represent summation of weighted basis functions. Framed insets show plots of residuals.
set, the superscript refers to the holoform of the protein, and n is a charge state of a protein ion. A χ2-minimized fit of the experimental data with a normal distribution is shown in Figure 2A. Although selection of Gaussian distribution as a mathematical representation of a basis function is arbitrary, it does seem to provide results that clearly have physical meaning (vide infra).
At pH 4.0, the charge-state distribution of hMb ions extends to higher charges, indicating the presence of alternative state(s) in addition to the native one represented by Bh1 (n). Deconvolution of this distribution has been done by assuming that the total number of basis functions is two and using the initial parameters for one of them as those for Bh1 (n) at pH 7.4. Charge states +13 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
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and higher have been neglected in this analysis, as the relative intensities of corresponding peaks do not exceed 3%. In the course of deconvolution, we were seeking to find the best fit between the calculated distribution Icalc(n) ) ∑biBhi (n) and the experimentally measured charge-state distribution of hMb ions Ih(n). Optimization was carried out using Origin’s standard Simplex routine by varying parameters both parameters (νio and σi) and a “weighting” coefficient bi for each basis function to minimize χ2. The results of this analysis are shown on Figure 2B (shaded curves). Charge-state distributions of the aMb ions observed in the spectra of hMb (pH 4.0 and below) and aMb (pH 2.5 to 8.6) were deconvoluted in a similar way. As an initial approximation, B1a(n) and B2a(n) were chosen in each case as Bh1 (n) and B2h(n). Initially, we have attempted to fit the observed distributions of aMb ions using only three basis functions. However, we were not able to find good fits even when any restrictions on B1a(n) and B2a(n) were completely removed. This problem was easily overcome by the expanding the set of basis functions to four. An independent analysis of the entire data set of aMb using an SVD algorithm30 has yielded four significant singular values, indicating that a self-sufficient set of basis functions should include four functions. The results of deconvolution of some of the charge-state distributions using four basis functions are shown in Figure 2BF. Remarkably (although not unexpectedly), the set of basis functions obtained in the course of the analysis does not show any significant variation over the entire pH range 2.5-8.6. Thus, the population central νio for each normal distribution representing basis functions for aMb remains constant across the entire pH range and independent of the presence of the heme group in solution (Figure 3 A,D). This supports our initial assumption that each basis function represents a single conformational state of the protein. Several earlier studies have concluded that acid unfolding of myoglobin in the “medium-salt” aqueous solution gives rise to four different states of the protein (in the order of decreased folding): native (N), so-called “pH 4 intermediate” (I), extended conformation (E), and unfolded state (U) (see, for example, recent work by Callender’s group31 and references therein). As the average charge state of each conformer (i.e., population central of a corresponding normal distribution) is an indicator of its overall compactness, assignment of the basis functions is very straightforward (i.e., B1a(n) for N, B2a(n) for I, B3a(n) for E, and B4a(n) for U). Another parameter that defines a basis function (in addition to the population central, νio) is the population variance, σi2. For the purpose of simplicity, we consider square roots of each variance (i.e., standard deviations of the normal distributions Bia(n), corresponding to the spread of each Gaussian curve). Standard deviations of each of the four basis functions Bia(n) do not exhibit significant variations as a function of pH and are also independent of the presence of the heme group in the solution (Figure 3 B,E). The order of σi suggests increasing conformational heterogeneity in the series of aMb states: N < I , E ∼ U, which (30) Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical recipies in C. The art of scientific computing.; Cambridge University Press: New York, 1988. (31) Gilmanshin, R.; Gulotta, M.; Dyer, R. B.; Callender, R. H. Biochemistry 2001, 40, 5127-5136.
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is in a good agreement with the results of the earlier studies based on spectroscopic measurements.31,32 Normalized values of σi (i.e., σi/νio) are also the lowest for the N state of the protein, followed by that of the I state across the entire pH range. Unlike population centrals and standard deviations, the “weighting” coefficients bi (i.e., relative contributions of the basis functions to the overall charge-state distribution of aMb) vary significantly as a function of pH (Figure 3 C,F). In the case of aMb, the relative abundances of all four conformers remain fairly constant at pH 5 and higher, with the highest ionic signal provided by N. This is followed by a rapid decrease of the relative ionic signal of N in the pH range 4.0-5.0. The relative ionic signals of the I and E conformers reach their respective maximums at pH 4.0-4.5 and 3.0-3.5. Finally, the relative ionic signal corresponding to the unfolded state of aMb (U) remains at constant and low level until the pH is lowered to 3, at which time its abundance begins to increase very dramatically. Characteristics of aMb and hMb Conformational Isomers Deduced from ESI MS. In the preceding section we have assigned the four basis functions obtained in the course of deconvolution protein ion charge-state distributions to the four different conformational states of aMb in solution (N, I, E, U). Since the acid unfolding of aMb has been thoroughly studied in recent years using a variety of techniques, it would be instructive to see if there is any correlation between the documented behavior of aMb equilibrium unfolding intermediates and the aMb conformers detected in the present work (as represented by the basis functions). The holoform of Mb is organized into eight R-helical segments (usually termed A-H).20 Removal of the heme group at neutral pH leads to a loss of structure in the region corresponding to the EF loop, F helix, FG loop, and part of the G helix, while the rest of the polypeptide chain spends most of the time in the “nativelike” conformation N, as suggested by multidimensional heteronuclear NMR measurements.33 This is consistent with lower helicity of aMb, as measured by CD spectroscopy,34 as well as its increased radius of gyration (19-20 Å vs ∼17.5 Å for hMb), as measured by SAXS.35,36 Lowering the solution pH results in accumulation of the intermediate form I (often termed “pH 4 intermediate”). The protein has a “looser” structure under these conditions, as suggested by the increased radius of gyration (2325 Å)35 and decreased helicity, although it is believed to retain a tight core (intersection of AGH helices) surrounded by partially solvated helical regions.37 Further decrease of pH leads to accumulation of yet another intermediate form of the protein, termed E (or “extended form”). This form is thought to contain the AGH core, whose size is significantly smaller, as compared to that of I or N, with only 24% of the protein existing in the “helical-like” environment.31 Interestingly, some residual helical structure and tertiary contacts are still detected at pH as low as 1.5, as suggested by UV resonance Raman spectroscopy32 and farUV CD measurements,31 leading to a suggestion that the acid (32) (33) (34) (35)
Chi, Z.; Asher, S. A. Biochemistry 1999, 38, 8196-8203. Elieser, D.; Wright, P. E. J. Mol. Biol. 1996, 263. Hirst, J. D.; Brooks, C. L., 3rd. J. Mol. Biol. 1994, 243, 173-178. Kataoka, M.; Nishii, I.; Fujisawa, T.; Ueki, T.; Tokunaga, F.; Goto, Y. J. Mol. Biol. 1995, 249, 215-228. (36) Eliezer, D.; Jennings, P. A.; Wright, P. E.; Doniach, S.; Hodgson, K. O.; Tsuruta, H. Science 1995, 270, 487-488. (37) Callender, R. H.; Dyer, R. B.; Gilmanshin, R.; Woodruff, W. H. Annu. Rev. Phys. Chem. 1998, 49, 173-202.
Figure 3. Characterization of Mb conformational isomers in solutions of hMb (A-C) and aMb (D-F) in the pH range 2.5-8.6: conformers’ sizes as represented by ion charge state (A, D), heterogeneity (B, E), and relative abundance of ionic signal (C, F). Mb states are denoted as follows: native hMb, B1h(n) (9); hMb, B2h(n) (0); native aMb, N (O); pH 4 intermediate state of aMb, I (b); extended state of aMb, E (2); and unfolded aMb, U (]).
“unfolded” state of the protein U also contains some residual helical structure. Acid unfolding intermediates I and E are known to be stabilized at “medium-salt” (
