Biomedical subjects

Carol V Robinson

Publications and source records attributed to Carol V Robinson.

At least 19 recordsLinked to original sources

PubMed2025

Uncovering hidden protein modifications with native top-down mass spectrometry.

Protein modifications drive dynamic cellular processes by modulating biomolecular interactions, yet capturing these modifications within their native structural context remains a significant challenge. Native top-down mass spectrometry promises to preserve the critical link between modifications and interactions. However, current methods often fail to detect uncharacterized or low-abundance modifications, limiting insights into proteoform diversity. To address this gap, we introduce precise and accurate Identification Of Native proteoforms (precisION), an interactive end-to-end software package that leverages a robust, data-driven fragment-level open search to detect, localize and quantify 'hidden' modifications within intact protein complexes. Applying precisION to four therapeutically relevant targets-PDE6, ACE2, osteopontin (SPP1) and a GABA transporter (GAT1)-we discover undocumented phosphorylation, glycosylation and lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map of GAT1. As an open-source software package, precisION offers an intuitive means for interpreting complex protein fragmentation data. This tool will empower the community to unlock the potential of native top-down mass spectrometry, advancing integrative structural biology, molecular pathology and drug development.

Mass Spectrometry↗

PubMed2006

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Animals↗

PubMed2006

Functional and structural characterization of the Methanosarcina mazei proteasome and PAN complexes.

We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon Methanosarcina mazei and produced the respective proteins in Escherichia coli cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by Methanosarcina proteasomes was activated by Methanosarcina PAN. Notably, the Methanosarcina PAN unfolded GFP-SsrA only in the presence of Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15 nm height and 11 nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18 nm and sixfold-symmetric top-views with a diameter of 15 nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the Methanosarcina and the Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon M. mazei and described unique new structural and functional features.

Adenosine Triphosphatases↗

PubMed2006

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.

Bacteriophage T4↗

PubMed2006

Mass spectrometry of macromolecular assemblies: preservation and dissociation.

Mass spectrometry not only plays a crucial role in the identification of proteins involved in the intricate interaction networks of the cell, but also is increasingly involved in the characterization of the non-covalent complexes formed by interacting partners. Recent developments have enabled the use of gas phase dissociation to probe oligomeric organization and topology, and increased understanding of the electrospray process is leading to knowledge of the structure of protein assemblies both in solution and in the gas phase.

Multiprotein Complexes↗

PubMed2006

Structural basis for the inhibition of activin signalling by follistatin.

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 A resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF- and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-beta family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-beta family proteins.

Activin Receptors, Type II↗

PubMed2006

Multimers of the fibroblast growth factor (FGF)-FGF receptor-saccharide complex are formed on long oligomers of heparin.

The minimal signalling unit for tyrosine kinase receptors is two protomers dimerized by one or more ligands. However, it is clear that maximal signalling requires the formation of larger complexes of many receptors at discrete foci on the cell surface. The biological interactions that lead to this are likely to be diverse and have system specific components. In the present study, we demonstrate that, in the FGF (fibroblast growth factor)-FGFR (FGF receptor) system, multimers of the minimal complex composed of two FGF1 and two FGFR2 protomers can form on a single chain of the co-receptor heparin. Using size-exclusion chromatography, we show that two complexes can form on heparin chains as small as 16 saccharide units. We also show by MS that discrete complexes containing exactly two copies of the minimal signalling unit are formed. However, the doublet of complexes appears to be less co-operative than the formation of the 2:2:1 FGF1:FGFR2:heparin complex, suggesting that this mechanism is one of a number of weaker interactions that might be involved in the formation of a focal complex on the cell surface.

Animals↗

PubMed2006

20S proteasomes have the potential to keep substrates in store for continual degradation.

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.

Cytochromes c↗

PubMed2006

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris.

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

Amyloidosis↗

PubMed2006

Regional and segmental flexibility of antibodies in interaction with antigens of different size.

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.

Animals↗

PubMed2006

Insights into amyloid fibril formation from mass spectrometry.

Mass spectrometry has become increasingly important in amyloid research specifically in the mechanism of formation and characterization of fibrils. In this review we highlight key experiments that provide evidence for different conformations, interactions and unfolding intermediates in proteins associated with amyloid diseases.

Amyloid↗

PubMed2005

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins.

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.

Adenosine Triphosphatases↗

PubMed2005

Evidence for macromolecular protein rings in the absence of bulk water.

We have examined the architecture of a protein complex in the absence of bulk water. By determining collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topology of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addition of a specific RNA molecule increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies.

5' Untranslated Regions↗

PubMed2005

L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers.

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

Amyloid↗

PubMed2005

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

Amyloidosis↗

PubMed2005

Protein complexes of the Escherichia coli cell envelope.

Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.

Blotting, Western↗

PubMed2005

Molecular recycling within amyloid fibrils.

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of beta-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

Amyloid↗

PubMed2005

Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis.

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles.

Amino Acid Motifs↗