Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta)

Abstract

Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.

Fig. 1.

Amino acid sequence of rhM-CSFβ with the peptic fragments used in this study. Cysteine residues are shaded. The α-helices and β strands are illustrated based on the X-ray structure of rhM-CSFα (Pandit et al. 1992).

Fig. 2.

ESI mass spectra with deconvoluted spectra (insets) of undeuterated rhM-CSFβ (A) and fully deuterated rhM-CSFβ (B).

Fig. 3.

(A) Mass spectra for singly and doubly charged ions of peptic peptide amino acids 37–49 at various H/D exchange times. The deuterium level in this fragment was deduced from centriods of the envelope isotope peaks of both ions, averaged and corrected with two reference samples. (B) Fitted curves of representative peptic fragments according to equation 1. Data for fragment of amino acids 190–221 was not able to deconvolute by equation 1.

Fig. 4.

Tandem mass spectrum of the doubly charged ion at m/z 1357.2 representing peptic peptide amino acids 114–135 showed a series of b_n ions. The b₁₅ ion was used to analyze the b₁–b₁₅ fragment. The deuterium level in this fragment was deduced from centriods of the envelope isotope peaks and was adjusted with m_0% and m_100% control samples (Zhang and Smith 1993).

Fig. 5.

A ribbon plot of rhM-CSFβ based on the crystal structure of rhM-CSFα (Pandit et al. 1992). The disulfide bonds were labeled and shown by stick model. The gray color shows amino acids not identified in the digest. Dotted lines represent the carboxy-terminal strands for which there are no X-ray data. The different colors indicate the regions with fast or intermediate H/D exchange rates (red), nonexchangeable or slow exchange rates (blue), or multiple categories of exchange rates (yellow). Average depths of the chains were obtained from X-ray coordinates.

Fig. 6.

The solvent accessibility percentage in each fragment, which is derived from the total number of incorporated deuteriums for fast, intermediate, and slow exchange (column 9 in Table 1) over the number of theorectically exchangeable amide hydrogens, correlated with the average depth for each fragment. The errors are the standard deviation for the average depth. The line through the data was calculated using a weighted least squares fit when each data point was weighted by 1/σ² and yielded the linear relationship of y = −0.017x + 6.604 (R = 0.88). The data used for this correlation calculation are the independent fragments with entry numbers from 1 to 19, and 24 in Table 1.

Fig. 7.

Comparison of a number of amino acid residues with different solvent exposures in the crystal structure (x-axes) in rhM-CSFα with a number of amide hydrogens having different H/D exchange rates (y-axes) in rhM-CSFβ. The number of amino acids that are highly exposed are compared to the number of amide hydrogens in fast exchange (A), partially exposed compared to amide hydrogens in intermediate exchange (B), and buried compared to amide hydrogens in slow or no exchange (C) for each independent peptide fragment listed in Table 1 (entry numbers 1 to 19, and 24). The least squares fit for the data in each panel yielded linear relationships of y = 0.77x − 1.33 (R = 0.79) for A, y = 1.18x − 0.46 (R = 0.83) for B, and y = 1.25x + 0.155 (R = 0.74) for C. Fragments 17, 18, and 19 are labeled in each panel.

Fig. 8.

Molecular model of the two subunits of rhM-CSFα. Labeled fragments in one subunit, but same color in other subunit, are shown as ribbon plots. The rest of the protein (gray area) is shown as space-filling model. Colored ribbons represent partial sequences in αB, αC, and αD helices.