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1.
Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry 总被引:8,自引:0,他引:8
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Nettleton EJ Tito P Sunde M Bouchard M Dobson CM Robinson CV 《Biophysical journal》2000,79(2):1053-1065
The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils. 相似文献
2.
Shiori Nakazawa Joomi Ahn Noritaka Hashii Kenji Hirose Nana Kawasaki 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(6):1210-1214
Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3–6 and B22–24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3–6, whereas they increased in B22–24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology. 相似文献
3.
McCammon MG Scott DJ Keetch CA Greene LH Purkey HE Petrassi HM Kelly JW Robinson CV 《Structure (London, England : 1993)》2002,10(6):851-863
Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein. 相似文献
4.
Protein modification by ubiquitin is a central regulatory mechanism in eukaryotic cells. Recent proteomics developments in mass spectrometry enable systematic analysis of cellular components in the ubiquitin pathway. Here, we review the advances in analyzing ubiquitinated substrates, determining modified lysine residues, quantifying polyubiquitin chain topologies, as well as profiling deubiquitinating enzymes based on the activity. Moreover, proteomic approaches have been developed for probing the interactome of proteasome and for identifying proteins with ubiquitin-binding domains. Similar strategies have been applied on the studies of the modification by ubiquitin-like proteins as well. These strategies are discussed with respect to their advantages, limitations and potential improvements. While the utilization of current methodologies has rapidly expanded the scope of protein modification by the ubiquitin family, a more active role is anticipated in the functional studies with the emergence of quantitative mass spectrometry. 相似文献
5.
Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products. 相似文献
6.
Brock A 《Protein expression and purification》2012,84(1):19-37
The ability of proteins to function is intricately connected to proper folding and other environmental parameters. Methods and tools that are able to report back on structure and dynamics in native or otherwise desired environment are of utmost importance as they can be used to connect structure and function and provide us with deeper mechanistic understanding of the underlying principles involved. Besides, they might be useful in the verification of material quality and provide assurance of efficacy and soundness of experimental observations made with such materials. Hydrogen exchange mass spectrometry is one of those tools. It combines the universality of a "native" chemical labeling approach with an almost equally universally applicable detection scheme based on mass spectrometry that has already revolutionized proteomic research as a whole in the past. This review focuses on the concepts of the exchange method, advances in methodology that have made specifically the fragmentation hydrogen exchange mass spectrometry approach so successful, broadly surveys some of the application space, and highlights the most recent use in the area of biopharmaceutical development. 相似文献
7.
《Biophysical journal》2022,121(23):4505-4516
Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formed β-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1–11, 1–15) and central (12–15, 16–25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25–30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation. 相似文献
8.
He J Song Y Ueyama N Saito A Azakami H Kato A 《Protein science : a publication of the Protein Society》2006,15(2):213-222
To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106. 相似文献
9.
Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating. 相似文献
10.
Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers. 相似文献
11.
Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. 总被引:18,自引:13,他引:5
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A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins. 相似文献
12.
The [Het-s] infectious element of the filamentous fungus Podospora anserina corresponds to the prion form of the HET-s protein. HET-s (289 amino acids in length) aggregates into amyloid fibers in vitro. Such fibers obtained in vitro are infectious, indicating that the [Het-s] prion can propagate as a self-perpetuating amyloid aggregate of the HET-s protein. Previous analyses have suggested that only a limited region of the HET-s protein is involved in amyloid formation and prion propagation. To document the conformational transition occurring upon amyloid aggregation of HET-s, we have developed a method involving hydrogen/deuterium exchange monitored by MALDI-MS. In a first step, a peptide mass fingerprint of the protein was obtained, leading to 87% coverage of the HET-s primary structure. Amyloid aggregates of HET-s were obtained, and H/D exchange was monitored on the soluble and on the amyloid form of HET-s. This study revealed that in the soluble form of HET-s, the C-terminal region (spanning from residues 240-289) displays a high solvent accessibility. In sharp contrast, solvent accessibility is drastically reduced in that region in the amyloid form. H/D exchange rates and levels in the N-terminal part of the protein (residues 1-220) are comparable in the soluble and the aggregated state. These results indicate that amyloid aggregation of HET-s involves a conformational transition of the C-terminal part of the protein from a mainly disordered to an aggregated state in which this region is highly protected from hydrogen exchange. 相似文献
13.
Lee T Hoofnagle AN Kabuyama Y Stroud J Min X Goldsmith EJ Chen L Resing KA Ahn NG 《Molecular cell》2004,14(1):43-55
Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions. 相似文献
14.
Wang L Lane LC Smith DL 《Protein science : a publication of the Protein Society》2001,10(6):1234-1243
Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D(2)O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N- and C-terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids. 相似文献
15.
Nazabal A Maddelein ML Bonneu M Saupe SJ Schmitter JM 《The Journal of biological chemistry》2005,280(14):13220-13228
The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops. 相似文献
16.
In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes. 相似文献
17.
18.
William Burkitt Paula Domann Gavin O'Connor 《Protein science : a publication of the Protein Society》2010,19(4):826-835
Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the CH2 and CH3 domains. 相似文献
19.
Li J Lim MS Li S Brock M Pique ME Woods VL Craig L 《Structure (London, England : 1993)》2008,16(1):137-148
The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens. 相似文献