A hydrophobic ratchet entrenches molecular complexes.

Most proteins assemble into multisubunit complexes1. The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work2. In many complexes, however, multimerization does not enable any known function3. An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function3-7. Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous.

Structure and mechanism of human diacylglycerol O-acyltransferase 1.

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.

Origin of complexity in haemoglobin evolution.

Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and ß-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and ß-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.

Author Correction: Origin of complexity in haemoglobin evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNß after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.

Structures of Get3d reveal a distinct architecture associated with the emergence of photosynthesis.

Homologs of the protein Get3 have been identified in all domains yet remain to be fully characterized. In the eukaryotic cytoplasm, Get3 delivers tail-anchored (TA) integral membrane proteins, defined by a single transmembrane helix at their C terminus, to the endoplasmic reticulum. While most eukaryotes have a single Get3 gene, plants are notable for having multiple Get3 paralogs. Get3d is conserved across land plants and photosynthetic bacteria and includes a distinctive C-terminal α-crystallin domain. After tracing the evolutionary origin of Get3d, we solve the Arabidopsis thaliana Get3d crystal structure, identify its localization to the chloroplast, and provide evidence for a role in TA protein binding. The structure is identical to that of a cyanobacterial Get3 homolog, which is further refined here. Distinct features of Get3d include an incomplete active site, a "closed" conformation in the apo-state, and a hydrophobic chamber. Both homologs have ATPase activity and are capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis suggesting a role in the homeostasis of photosynthetic machinery.

Nonlinear Frequency Modulation for Fourier Transform Ion Mobility Mass Spectrometry Improves Experimental Efficiency.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Cardiolipin Regulates the Activity of the Mitochondrial ABC Transporter ABCB10.

The ATP-binding cassette (ABC) transporter ABCB10 resides in the inner membrane of mitochondria and is implicated in erythropoiesis. Mitochondria from different cell types share some specific characteristics, one of which is the high abundance of cardiolipin. Although previous studies have provided insight into ABCB10, the affinity and selectivity of this transporter toward lipids, particularly those found in the mitochondria, remain poorly understood. Here, native mass spectrometry is used to directly monitor the binding events of lipids to human ABCB10. The results reveal that ABCB10 binds avidly to cardiolipin with an affinity significantly higher than that of other phospholipids. The first three binding events of cardiolipin display positive cooperativity, which is suggestive of specific cardiolipin-binding sites on ABCB10. Phosphatidic acid is the second-best binder of the lipids investigated. The bulk lipids, phosphatidylcholine and phosphatidylethanolamine, display the weakest binding affinity for ABCB10. Other lipids bind ABCB10 with a similar affinity. Functional assays show that cardiolipin regulates the ATPase activity of ABCB10 in a dose-dependent fashion. ATPase activity of ABCB10 was also impacted in the presence of other lipids but to a lesser extent than cardiolipin. Taken together, ABCB10 has a high binding affinity for cardiolipin, and this lipid also regulates the ATPase activity of the transporter.

2'-Deoxy Guanosine Nucleotides Alter the Biochemical Properties of Ras.

Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-RasG12C was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOScat) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOScat differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.

Selective regulation of human TRAAK channels by biologically active phospholipids.

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

Surface Activity of Amines Provides Evidence for the Combined ESI Mechanism of Charge Reduction for Protein Complexes.

Charge reduction reactions are important for native mass spectrometry (nMS) because lower charge states help retain native-like conformations and preserve noncovalent interactions of protein complexes. While mechanisms of charge reduction reactions are not well understood, they are generally achieved through the addition of small molecules, such as polyamines, to traditional nMS buffers. Here, we present new evidence that surface-active, charge reducing reagents carry away excess charge from the droplet after being emitted due to Coulombic repulsion, thereby reducing the overall charge of the droplet. Furthermore, these processes are directly linked to two mechanisms for electrospray ionization, specifically the charge residue and ion evaporation models (CRM and IEM). Selected protein complexes were analyzed in solutions containing ammonium acetate and selected trialkylamines or diaminoalkanes of increasing alkyl chain lengths. Results show that amines with higher surface activity have increased propensities for promoting charge reduction of the protein ions. The electrospray ionization (ESI) emitter potential was also found to be a major contributing parameter to the prevalence of charge reduction; higher emitter potentials consistently coincided with lower average charge states among all protein complexes analyzed. These results offer experimental evidence for the mechanism of charge reduction in ESI and also provide insight into the final stages of the ESI and their impact on biological ions.

Step toward Probing the Nonannular Belt of Membrane Proteins.

Integral membrane proteins are embedded in the biological membrane, where they carry out numerous biological processes. Although lipids present in the membrane are crucial for membrane protein function, it remains difficult to characterize many lipid binding events to membrane proteins, such as those that form the annular belt. Here, we use native mass spectrometry along with the charge-reducing properties of trimethylamine N-oxide (TMAO) to characterize a large number of lipid binding events to the bacterial ammonia channel (AmtB). In the absence of TMAO, significant peak overlap between neighboring charge states is observed, resulting in erroneous abundances for different molecular species. With the addition of TMAO, the weighted average charge state (Zavg) was decreased. In addition, the increased spacing between nearby charge states enabled a higher number of lipid binding species to be observed while minimizing mass spectral peak overlap. These conditions helped us to determine the equilibrium binding constants (Kd) for up to 16 lipid binding events. The binding constants for the first few lipid binding events display the highest affinity, whereas the binding constants for higher lipid binding events converge to a similar value. These findings suggest a transition from nonannular to annular lipid binding to AmtB.

Small molecule peptidomimetic trypsin inhibitors: validation of an EKO binding mode, but with a twist.

Examination of a series of naturally-occurring trypsin inhibitor proteins, led to identification of a set of three residues (which we call the "interface triplet") to be determinant of trypsin binding affinity, hence excellent templates for small molecule mimicry. Consequently, we attempted to use the Exploring Key Orientation (EKO) strategy developed in our lab to evaluate small molecules that mimic the interface triplet regions of natural trypsin inhibitors, and hence potentially might bind and inhibit the catalytic activity of trypsin. A bis-triazole scaffold ("TT-mer") was the most promising of the molecules evaluated in silico. Twelve such compounds were synthesized and assayed against trypsin, among which the best showed a Kd of 2.1 µM. X-ray crystallography revealed a high degree of matching between an illustrative TT-mer's actual binding mode and that of the mimics that overlaid the interface triplet in the crystal structure. Deviation of the third side chain from the PPI structure seems to be due to alleviation of an unfavorable dipole-dipole interaction in the small molecule's actual bound conformation.

Insight into the Phospholipid-Binding Preferences of Kir3.4.

The G-protein-gated inwardly rectifying potassium channel 4 (Kir3.4) subunit forms functional tetramers. Previous studies have established that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required for Kir3.4 function. However, the binding preferences of Kir3.4 for the headgroup and acyl chains of phosphorylated phosphatidylinositides (PIPs) and other lipids are not well understood. Here, the interactions between full-length, human Kir3.4 and lipids are characterized using native mass spectrometry (MS) in conjunction with a soluble fluorescent lipid-binding assay. Kir3.4 displays binding preferences for PIPs, and, in some cases, the degree of binding is influenced by the type of acyl chains. The interactions between Kir3.4 and PIPs are weaker in comparison to full-length, human Kir3.2. The binding of PI(4,5)P2 modified with a fluorophore to Kir3.2 can be enhanced by other lipids, such as phosphatidylcholine. Introduction of S143T, a mutation that enhances Kir3.4 activity, results in an overall reduction in the channel binding PIPs. In contrast, the D223N mutant of Kir3.4 that mimics the sodium-bound state exhibited stronger binding for PI(4,5)P2, particularly for those with 18:0-20:4 acyl chains. Taken together, these results provide additional insight into the interaction between Kir3.4 and lipids that are important for channel function.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding.

Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.