Native Mass Spectrometry Captures the Conformational Plasticity of Proteins with Low-Complexity Domains

Abstract

Disordered regions are an important functional feature of many multidomain proteins. A prime example is proteins in membraneless organelles, which contain folded domains that engage in specific interactions and disordered low-complexity (LC) domains that mediate liquid-liquid phase separation. Studying these complex architectures remains challenging due to their conformational variability. Native mass spectrometry (nMS) is routinely employed to analyze conformations and interactions of folded or disordered proteins; however, its ability to analyze proteins with disordered LC domains has not been investigated. Here, we analyze the ionization and conformational states of designed model proteins that recapitulate key features of proteins found in membraneless organelles. Our results show that charge state distributions (CSDs) in nMS reflect partial disorder regardless of the protein sequence, providing insights into their conformational plasticity and interactions. By applying the same CSD analysis to a spider silk protein fragment, we find that interactions between folded domains that trigger silk assembly simultaneously induce conformational changes in the LC domains. Lastly, using intact nucleosomes, we demonstrate that CSDs are a good predictor for the disorder content of complex native assemblies. We conclude that nMS reliably informs about the conformational landscape of proteins with LC domains, which is crucial for understanding protein condensates in cellular environments.

Figure 1

Design of partially folded proteins with disordered LC domains. (a) FUS and TDP-43 are examples of proteins that undergo LLPS via LC domains that are virtually devoid of charged residues. (b) Overall architecture of the model proteins shown as FuzDrop score (left) and AF3 model (right). The NT* domain (teal) scores low for LLPS propensity and is connected to a 70-residue LC domain with high LLPS propensity (mint). The locations of residues with positive (blue) and negative (red) solution charge are indicated in the gray bars above the FuzDrop plots. (c) FuzDrop scores of the (GSGAP)₁₄, (GSGAY)₁₄, (GSGAE)₁₄, and (GSGAK)₁₄ repeats show high LLPS propensity. (d) IDPGan ensembles of ten copies of each repeat show complete disorder and a slightly lower degree of compaction for the (GSGAE)₁₄ and (GSGAK)₁₄ repeats. The NT* domain (blue) is shown for scale in the left panel. (e) Light microscopy images of 20 μM NT*-LC proteins in 100 mM ammonium acetate, pH 8, show no droplet formation. NT-2Rep droplets formed in 0.75 M phosphate buffer are shown as positive control on the far right. Scale bars are 10 μm.

Figure 2

Charge states of partially folded model proteins with LC domains reflect relative disorder content and surface area. (a) Representative native mass spectra for NT*-(GSGAP)₁₄, NT*-(GSGAY)₁₄, NT*-(GSGAE)₁₄, and NT*-(GSGAK)₁₄ (top to bottom) show trimodal CSDs. The highest charge state envelope is highlighted in blue and centered on 17+, 14+, 16+, and 17+, respectively, while the intermediate envelope (purple) and the lowest envelope (pink) are centered on 10+ and 8+. Asterisks indicate nonprotein peaks. (b) Predicting the charge of a protein with a globular (teal) and a disordered domain (mint). The mass of the disordered domain is 25% of that of the whole protein, which is the case for NT-LC proteins used here. The expected charge of each domain is calculated separately using the empirical formulas for either compact (i) or disordered proteins (ii). The expected charges are then summed to predict the total charge for a protein in which both domains are disordered, the LC domain is disordered, both domains are compacted separately, or both domains are compacted together. (c) Comparison of the predicted and experimental charge states for partially disordered proteins. Expected average charges as a function of molecular weight are shown as solid lines for fully disordered and for completely folded proteins. Dashed lines indicate the expected average charge for a protein with an extended disordered domain or a compact disordered domain. The average charges of the three CDSs for each of the NT*-LC proteins are shown using the same color code as in (a) and correspond to a compact and an unfolded domain (blue), two compact domains (purple), and a single collapsed protein (pink).

Figure 3

Disordered regions without positively charged residues ionize using NH₄⁺ as charge carriers and retain protons upon collisional activation. (a) Representative native mass spectra of NT*-(GSGAP)₁₄, NT*-(GSGAY)₁₄, and NT*-(GSGAE)₁₄ show a mixture of charge carriers. Each charge state has five protons and a variable number of ammonium ions. Higher charge states (>9+) also exist as an isolated peak with only protons as charge carriers. For NT*-(GSGAK)₁₄, which contains an excess of residues with a positive solution charge, no ammonium adducts were observed for the same charge states. (b) Collisional activation results in the loss of ammonia adducts, leaving the proton-only peak.

Figure 4

nMS captures conformational changes in the LC domain of a spider silk protein. (a) NT-2Rep fragment contains the wild-type NT domain and two LC repeats with seven basic residues in total. The LC domain is predicted to undergo LLPS. The gray stripe above the graph indicates the location of residues with positive (blue) and negative solution charge (red). (b) AF3 structure prediction of the dimerized truncated NT-2Rep version, with plDDT score coloring of one subunit and a transparent second subunit behind it. (c) Representative native mass spectra of NT-2Rep at pH 8 (top) and pH 6 (bottom) show a broad CSD. At low pH, two dimer populations can be observed that are centered on the 21+ (blue) and 9+ (purple) charge states. (d) CSD predictions for NT-2Rep dimers with both LC domains extended, one LC domain collapsed, and both LC domains collapsed show that the high and low experimental CSDs agree with two extended and two collapsed LC domains, respectively.

Figure 5

CSDs indicate the extent of disorder in intact nucleosomes. (a) Native mass spectra for nucleosomes show trimodal CSDs. The highest charge state envelope is highlighted in blue and centered on 43+, while the intermediate envelope (purple) and the lowest envelope (pink) are centered on 35+ and 29+, respectively. (b) Empirical calculations for the average charge as a function of molecular weight are shown for a protein that contains 10% disordered and 90% ordered (upper dashed line) or 5% disordered and 95% ordered region of the total molecular weight (middle dashed line) and completely collapsed state (lower dashed line). The average charges of each of the three CDSs are shown using the same color code as in (a).