Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,3'-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47 degrees C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.     

Structure and expression of a yeast gene encoding the small heat-shock protein Hsp26

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding a small heat-shock protein (Hsp26) has been determined. It reveals a 213-amino acid protein (27 kDa) that contains no methionine (Met) residues. Radiolabelling studies demonstrate the N-terminal Met residue is cleaved post-translationally. The Hsp26 amino acid sequence shows significant homology with both a range of eukaryotic small Hsps and with vertebrate alpha-crystallins. Particularly highly conserved among these proteins is a hydrophobic tetrapeptide sequence Gly-Val-Leu-Thr. These findings are discussed in relation to the structure and function of small Hsps.     

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N‐ and C‐terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast‐localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N‐terminal arms located on the outside of the dodecamer and six inwardly‐facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small‐angle X‐ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone‐client complexes were monitored and the R_g‐values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N‐terminal arms of the Hsp21 dodecamer. No such increase in R_g‐values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone‐client interactions involve the N‐terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N‐terminal arms of Hsp21 can trap and refold early‐unfolding intermediates with or without dodecamer dissociation.     

PKA phosphorylation of the small heat-shock protein Hsp20 enhances its cardioprotective effects

The small heat-shock protein Hsp20 (heat-shock protein 20), also known as HspB6, has been shown to protect against a number of pathophysiological cardiac processes, including hypertrophy and apoptosis. Following β-adrenergic stimulation and local increases in cAMP, Hsp20 is phosphorylated on Ser16 by PKA (protein kinase A). This covalent modification is required for many of its cardioprotective effects. Both Hsp20 expression levels and its phosphorylation on Ser16 are increased in ischaemic myocardium. Transgenic mouse models with cardiac-specific overexpression of Hsp20 that are subject to ischaemia/reperfusion show smaller myocardial infarcts, and improved recovery of contractile performance during the reperfusion phase, compared with wild-type mice. This has been attributed to Hsp20's ability to protect against cardiomyocyte necrosis and apoptosis. Phosphomimics of Hsp20 (S16D mutants) confer improved protection from β-agonist-induced apoptosis in the heart, whereas phospho-null mutants (S16A) provide no protection. Naturally occurring mutants of Hsp20 at position 20 (P20L substitution) are associated with markedly reduced Hsp20 phosphorylation at Ser16, and this lack of phosphorylation correlates with abrogation of Hsp20's cardioprotective effects. Therefore phosphorylation of Hsp20 at Ser16 by PKA is vital for the cardioprotective actions of this small heat-shock protein. Selective targeting of signalling elements that can enhance this modification represents an exciting new therapeutic avenue for the prevention and treatment of myocardial remodelling and ischaemic injury.     

Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q31 and the C-terminal K162 are involved in inter- and intramolecular crosslinking (transamidation). Q31 is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q66, which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.     

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.     

High resolution characterization of formamidopyrimidine-DNA glycosylase interaction with its substrate by chemical cross-linking and mass spectrometry using substrate analogs

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOgg1) initiate the base excision repair pathway for 7,8-dihydro-8-oxoguanine (8-oxoG) residues present in DNA. Recent structural and biochemical studies of Fpg-DNA and hOgg1-DNA complexes point to the existence of extensive interactions between phosphate groups and amino acids. However, the role of these contacts and their physiological relevance remains unclear. In the present study, we combined chemical cross-linking and electrospray ionization mass spectrometry (ESI/MS/MS) approaches to identify interacting residues in the Fpg-DNA and hOgg1-DNA complexes. The active centers of Fpg and hOgg1 were cross-linked with a series of reactive oligonucleotide duplexes containing both a single 8-oxoG residue and an O-ethyl-substituted pyrophosphate internucleotide (SPI) group at different positions in duplex DNA. The cross-linking efficiency reached 50% for Fpg and 30% for hOgg1. We have identified seven phosphate groups on both strands of the DNA duplex specifically interacting with nucleophilic amino acids in Fpg, and eight in hOgg1. MS/MS analysis of the purified proteolytic fragments suggests that lysine 56 of Fpg and lysine 249 of hOgg1 cross-link to the phosphate located 3' to the 8-oxoG residue. Site-specific mutagenesis analysis of Fpg binding to DNA substrate confirms the conclusions of our approach. Our results are consistent with crystallographic data on the Fpg-DNA complex and provide new data on the hOgg1-DNA interaction. The approach developed in this work provides a useful tool to study pro- and eukaryotic homologues of Fpg as well as other repair enzymes.     

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.     

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.     

Probing the transition-state structure of dual-specificity protein phosphatases using a physiological substrate mimic

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective timing of the proton transfer to the leaving group and P-O fission; with the alkyl ester substrate, protonation is ahead of P-O fission, while with the aryl substrate, the two processes are more synchronous. Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement of Asp-92 in protonating the ester oxygen, either in a step prior to significant P-O bond cleavage or in a concerted but asynchronous mechanism in which protonation is ahead of P-O bond fission. Collectively, the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of the proton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over with similar catalytic efficiency.     

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14?? was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19?? of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.     

Exploring the cooperativity of the fast folding reaction of a small protein using pulsed thiol labeling and mass spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.     

Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry

Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate-protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI-MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269-->Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C-3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H-bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral membrane proteins.     

Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.     

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry

To investigate protein–protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH–MutL–DNA complex.     

A partial set covering model for protein mixture identification using mass spectrometry data

Protein identification is a key and essential step in mass spectrometry (MS) based proteome research. To date, there are many protein identification strategies that employ either MS data or MS/MS data for database searching. While MS-based methods provide wider coverage than MS/MS-based methods, their identification accuracy is lower since MS data have less information than MS/MS data. Thus, it is desired to design more sophisticated algorithms that achieve higher identification accuracy using MS data. Peptide Mass Fingerprinting (PMF) has been widely used to identify single purified proteins from MS data for many years. In this paper, we extend this technology to protein mixture identification. First, we formulate the problem of protein mixture identification as a Partial Set Covering (PSC) problem. Then, we present several algorithms that can solve the PSC problem efficiently. Finally, we extend the partial set covering model to both MS/MS data and the combination of MS data and MS/MS data. The experimental results on simulated data and real data demonstrate the advantages of our method: 1) it outperforms previous MS-based approaches significantly; 2) it is useful in the MS/MS-based protein inference; and 3) it combines MS data and MS/MS data in a unified model such that the identification performance is further improved.