Janna Kiselar

Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the structure of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions using synchrotron light modify amino acid side-chains on millisecond timescales. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side-chains to the bulk solvent. The aromatic and sulphur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for a number of proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates correlate closely with a theoretical measure of the accessibility of residue side-chains to the solvent in the native protein structure. This approach can distinguish the relative accessibility of the tryptophan residue side-chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues, and phenylalanine at position 34 from phenylalanine residues at positions 3 and 38 based upon their rates of oxidation.

Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca2+-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca2+ concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca2+ sensitive. Ca2+titration isotherms monitoring the oxidation within residues 49–72 (within subdomain S1), 121–135 (S1), 162–166 (S2), and 722–748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca2+ concentration of 1–5 μM that is competent for capping and severing activity. A second structural transition with a midpoint of ≈60–100 μM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical “latch” of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca2+ transition. However, residues within S4 that are part of an extended β-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca2+ concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.

The Beamline X28C of the Center for Synchrotron Biosciences: a National Resource for Biomolecular Structure and Dynamics Experiments Using Synchrotron Footprinting

Structural mapping of proteins and nucleic acids with high resolution in solution is of critical importance for understanding their biological function. A wide range of footprinting technologies have been developed over the last ten years to address this need. Beamline X28C, a white-beam X-ray source at the National Synchrotron Light Source of Brookhaven National Laboratory, functions as a platform for synchrotron footprinting research and further technology development in this growing field. An expanding set of user groups utilize this national resource funded by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health. The facility is operated by the Center for Synchrotron Biosciences and the Center for Proteomics of Case Western Reserve University. The facility includes instrumentation suitable for conducting both steady-state and millisecond time-resolved footprinting experiments based on the production of hydroxyl radicals by X-rays. Footprinting studies of nucleic acids are routinely conducted with X-ray exposures of tens of milliseconds, which include studies of nucleic acid folding and their interactions with proteins. This technology can also be used to study protein structure and dynamics in solution as well as protein-protein interactions in large macromolecular complexes. This article provides an overview of the X28C beamline technology and defines protocols for its adoption at other synchrotron facilities. Lastly, several examples of published results provide illustrations of the kinds of experiments likely to be successful using these approaches.

Structural analysis of gelsolin using synchrotron protein footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron “white” beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its “inactive” and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.

Preservation and detection of specific antibody—peptide complexes by matrix-assisted laser desorption ionization mass spectrometry

The direct detection of an antibody—peptide complex is reported by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Experimental conditions have been found in which specific, noncovalent interactions in solution are maintained throughout the sample preparation and ionization process. Mass measurements based on the ion signals for the intact antibody and 1:1 antibody—peptide complex reveal that specific noncovalent associations between a monoclonal antibody and a peptide, which comprises the determinant of the corresponding antigen, are maintained in the gas phase. These results support the wider application of MALDI-MS to studies of the structure and specificity of macromolecular complexes important to immune and other biological function.

Visualizing Arp2/3 complex activation mediated by binding of ATP and wasp using structural mass spectrometry

Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprinting with mass spectrometry in solution to probe the effects of nucleotide- and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches. First, Arp2/3 is the most complex macromolecular assembly yet examined; second, only a few picomoles of Arp2/3 was required for individual experiments. In terms of structural biology of Arp 2/3, we find that ATP binding induces conformational changes within Arp2/3 complex in Arp3 (localized in peptide segments 5–18, 212–225, and 318–327) and Arp2 (within peptide segment 300–316). These data are consistent with nucleotide docking within the nucleotide clefts of the actin-related proteins promoting closure of the cleft of the Arp3 subunit. However, ATP binding does not induce conformational changes in the other Arp subunits. Arp2/3 complex binds to WASp within the C subdomain at residue Met 474 and within the A subdomain to Trp 500. Our data suggest a bivalent attachment of WASp to Arp3 (within peptides 162–191 and 318–329) and Arp2 (within peptides 66–80 and 87–97). WASp-dependent protections from oxidation within peptides 54–65 and 80–91 of Arp3 and in peptides 300–316 of Arp2 suggest domain rearrangements of Arp2 and Arp3 resulting in a closed conformational state consistent with an “actin-dimer” model for the active state.

Integrated Algorithms for High Throughput Examination of Covalently Labeled Biomolecules by Structural Mass Spectrometry

Mass spectrometry based structural proteomics approaches for probing protein structures are increasingly gaining in popularity. The potential for such studies is limited because of the lack of analytical techniques for the automated interpretation of resulting data. In this article, a suite of algorithms called ProtMapMS is developed, integrated, and implemented specifically for the comprehensive automatic analysis of mass spectrometry data obtained for protein structure studies using covalent labeling. The functions include data format conversion, mass spectrum interpretation, detection, and verification of all peptide species, confirmation of the modified peptide products, and quantification of the extent of peptide modification. The results thus obtained provide valuable data for use in combination with computational approaches for protein structure modeling. The structures of both monomeric and hexameric forms of insulin were investigated by oxidative protein footprinting followed by high-resolution mass spectrometry. The resultant data was analyzed both manually and using ProtMapMS without any manual intervention. The results obtained using the two methods were found to be in close agreement and overall were consistent with predictions from the crystallographic structure.

Ribosomal S6 Kinase 2 Directly Phosphorylates the 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptor, Thereby Modulating 5-HT2A Signaling

The 5-hydroxytryptamine 2A (5-HT2A) receptor is a member of the G protein-coupled receptor superfamily (GPCR) and plays a key role in transducing a variety of cellular signals elicited by 5-hydroxytryptamine in both peripheral and central tissues. Despite its broad physiological importance, our current understanding of 5-HT2A receptor regulation is incomplete. We recently reported the novel finding that the multifunctional ERK effector ribosomal S6 kinase 2 (RSK2) physically interacts with the 5-HT2A receptor third intracellular (i3) loop and modulates receptor signaling (Sheffler, D. J., Kroeze, W. K., Garcia, B. G., Deutch, A. Y., Hufeisen, S. J., Leahy, P., Bruning, J. C., and Roth, B. L. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 4717–4722). We report here that RSK2 directly phosphorylates the 5-HT2A receptor i3 loop at the conserved residue Ser-314, thereby modulating 5-HT2A receptor signaling. Furthermore, these studies led to the discovery that RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT2A receptor. We arrived at these conclusions via multiple lines of evidence, including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. Our findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies, and whole-cell signaling experiments. These results support a novel regulatory mechanism in which a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT2A serotonin receptor. To our knowledge, these findings are the first to demonstrate that a downstream member of the ERK/MAPK cascade phosphorylates a GPCR as well as mediates cross-talk between a growth factor and a GPCR.

Cellular Prion Protein Regulates Its Own α-Cleavage through ADAM8 in Skeletal Muscle

Background: Endoproteolytic α-cleavage of cellular prion protein (PrPC) regulates PrPC toxicity and functions; the responsible protease(s) is uncertain. Results: ADAM8 performs α-cleavage of PrPC and PrPC overexpression up-regulates ADAM8 in muscle. Conclusion: ADAM8 is the primary protease for the α-cleavage of PrPC that appears self-regulated through ADAM8 in muscle. Significance: This advances our understandings on physiological processing and functions of PrPC. The ubiquitously expressed cellular prion protein (PrPC) is subjected to the physiological α-cleavage at a region critical for both PrP toxicity and the conversion of PrPC to its pathogenic prion form (PrPSc), generating the C1 and N1 fragments. The C1 fragment can activate caspase 3 while the N1 fragment is neuroprotective. Recent articles indicate that ADAM10, ADAM17, and ADAM9 may not play a prominent role in the α-cleavage of PrPC as previously thought, raising questions on the identity of the responsible protease(s). Here we show that, ADAM8 can directly cleave PrP to generate C1 in vitro and PrP C1/full-length ratio is greatly decreased in the skeletal muscles of ADAM8 knock-out mice; in addition, the PrP C1/full-length ratio is linearly correlated with ADAM8 protein level in myoblast cell line C2C12 and in skeletal muscle tissues of transgenic mice. These results indicate that ADAM8 is the primary protease responsible for the α-cleavage of PrPC in muscle cells. Moreover, we found that overexpression of PrPC led to up-regulation of ADAM8, suggesting that PrPC may regulate its own α-cleavage through modulating ADAM8 activity.

Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine

Background: Chemokines oligomerize upon glycosaminoglycans to establish chemokine gradients. Results: Monomeric monocyte chemoattractant protein (MCP)-3/CCL7 has a dense network of glycosaminoglycan-binding epitopes that provide sufficient affinity for glycosaminoglycans, but the inability to oligomerize renders it sensitive to glycosaminoglycan density unlike the oligomerizing homolog, MCP-1/CCL2. Conclusion: Different glycosaminoglycan-binding properties of CCL7 and CCL2 suggest non-redundant functions and regulation. Significance: Glycosaminoglycan density may regulate the cell surface accumulation of chemokines. The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.