Chemistry Publications

Hydrogen Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Lars Konermann et al. — Mon, 30 May 2011 12:43:51 PDT

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.

Characterizing Short-Lived Protein Folding Intermediates by Top-Down Hydrogen Exchange Mass Spectrometry

Jingxi Pan et al. — Mon, 06 Dec 2010 19:02:20 PST

This work combines pulsed hydrogen/deuterium exchange (HDX) and top-down mass spectrometry for the structural characterization of short-lived protein folding intermediates. A custom-built flow device with three sequential mixing steps is used for (i) triggering protein folding, (ii) pulsed D(2)O labeling, and (iii) acid quenching. The earliest folding time point that can be studied with this system is 10 ms. The mixing device was coupled online to the electrospray source of a Fourier transform mass spectrometer, where intact protein ions are fragmented by electron capture dissociation (ECD). The viability of this experimental strategy is demonstrated by applying it to the refolding of horse apo-myoglobin (aMb), a reaction known to involve a transient intermediate. Cooling of the mixing device to 0 °C reduces the reaction rate such that the folding process occurs within the experimentally accessible time window. Top-down ECD provides an average spatial resolution of ca. 2 residues, surpassing the resolution typically achieved in traditional proteolytic digestion/HDX studies. Amide back exchange is virtually eliminated by the short (∼1 s) duration of the acid quenching step. The aMb folding intermediate exhibits HDX protection in helices G and H, whereas the remainder of the protein is largely unfolded. Marginal protection is seen for helix A. Overall, the top-down ECD approach used here offers insights into the sequence of events leading from the unfolded state to the native conformation, with envisioned future applications in the areas of protein misfolding and aggregation. The time-resolved experiments reported herein represent an extension of our previous work, where HDX/MS with top-down ECD was employed for monitoring "static" protein structures under equilibrium conditions (Pan et al. J. Am. Chem. Soc. 2009, 131, 12801).

Site-Directed Mutagenesis Combined with Oxidative Methionine Labeling for Probing Structural Transitions of a Membrane Protein by Mass Spectrometry

Yan Pan et al. — Sun, 21 Nov 2010 17:24:51 PST

Exposure of the membrane protein bacteriorhodopsin (BR) to SDS induces partial breakdown of the native conformation. The exact structural properties of this SDS state remain a matter of debate, despite its widespread use in BR folding experiments. The current work employs hydroxyl radical (·OH) labeling in conjunction with mass spectrometry (MS)-based peptide mapping for probing the solvent accessibility of individual BR segments in the presence of SDS. Previous work revealed methionine sulfoxide formation to be the dominant oxidative pathway. Those data suggested extensive unfolding of helices A and D in SDS. Unfortunately, the lack of Met residues in helices C and F implies that no direct information on the behavior of the latter two elements could be obtained. Here, we address this problem by employing two variants with additional Met residues, L93M (helix C) and V179M (helix F). The oxidation behavior of the resulting 11 methionines can be grouped into three categories: (1) extensively labeled both in native BR and in SDS (loop residues M32, M68, and M163); (2) protected in the native state but not in SDS (M20, M118); (3) always protected (M56, M60, M93, M145, M179, M209). These data show that a solvent-inaccessible core is retained in SDS. This core consists of partially intact helices B, C, E, F, and G. The termini of these helices are highly dynamic and/or unraveled, particularly on the cytoplasmic side. Overall, this work demonstrates how the use of engineered ·OH labeling sites can provide insights into structural properties of membrane proteins.

Synthesis of MRI Contrast Agents Derived from DOTAM-Gly-L-Phe-OH Incorporating a Disulfide Bridge: Conjugation to a Cell Penetrating Peptide and Preparation of a Dimeric Agent

Mojmír Suchý et al. — Mon, 20 Sep 2010 00:17:19 PDT

A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-Gly-L-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these agents have been evaluated.

Surface Charge of Electrosprayed Water Nanodroplets: A Molecular Dynamics Study

Elias Ahadi et al. — Tue, 07 Sep 2010 17:13:47 PDT

Aqueous nanodroplets that contain excess charge carriers play a central role during the electrospray ionization (ESI) process. An interesting question concerns the charge carrier location in these systems. In analogy to the behavior of metallic conductors, it is often assumed that excess ions are confined to a thin layer on the droplet surface. However, it is unclear whether simple electrostatic arguments adequately reflect the nanodroplet behavior. In particular, most ions tend to be heavily solvated, such that placing them at the liquid/vapor interface would be enthalpically unfavorable. In this work, molecular dynamics simulations are used to study the properties of Na(+)-containing water nanodroplets close to the Rayleigh limit. In apparent violation of the surface charge paradigm, it is found that the ions reside inside the droplet. Electrostatic mapping reveals that all of the excess charge is nonetheless located on the surface. This conundrum is resolved by considering the effects of orientational water polarization. Buried Na(+) ions cause large-scale dipole ordering that extends all the way to the droplet periphery. Here, the positive ends of water dipoles preferentially point into the vapor phase. These half-dipoles in the outermost droplet layers assume the role of surface charge, while solvation effectively neutralizes Na(+) ions in the interior. Overall, our data reaffirm the validity of the surface charge paradigm for ESI nanodroplets, albeit with the caveat that this paradigm does NOT require charge carriers (ions) to be located at the water/vapor interface.

RHAMM Promotes Interphase Microtubule Instability and Mitotic Spindle Integrity through MEK1/ERK1/2 Activity

Cornelia Tolg et al. — Wed, 18 Aug 2010 18:29:13 PDT

An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163-794 termed RHAMM(Delta163)) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMM(Delta163) modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM(-/-) mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMM(Delta163) binds to alpha- and beta-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMM(Delta163), ERK1/2-MEK1, and alpha- and beta-tubulin and demonstrate direct binding of RHAMM(Delta163) to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMM(Delta163) defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM(Delta163) on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMM(Delta163) functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.

Laminar Flow Effects During Laser-Induced Oxidative Labeling for Protein Structural Studies by Mass Spectrometry

Lars Konermann et al. — Sat, 07 Aug 2010 13:58:49 PDT

Laser-induced oxidative labeling of proteins provides insights into biomolecular structures and interactions. In these experiments, the hydroxyl radical ((*)OH) formed by photolysis of H(2)O(2) generates covalent modifications that are detectable by mass spectrometry. Under conditions where individual protein molecules are irradiated only once, the short (*)OH lifetime ( approximately 1 mus) ensures that covalent modifications are formed before any oxidation-induced conformational changes take place. This feature implies that the method should be free of structural artifacts. It has been proposed that single-exposure conditions can be achieved by passing the solution through a capillary where successive laser pulses generate a string of irradiated flow segments that are well separated from one another. The current work explores the convection phenomena within the labeling capillary in more detail. The experiments are conducted at Reynolds numbers <<2000, resulting in laminar flow. The associated parabolic velocity profile causes a portion of each irradiated segment to remain in the labeling window during the subsequent laser pulse. Achieving a genuine single-exposure regime is, therefore, not possible. We estimate the fraction of labeled protein formed under laminar flow conditions, as well as the occurrence of multiple exposure events for any combination of experimental parameters (laser spot width, pulse frequency, and solution flow rate). A proper choice of these parameters provides extensive labeling, while keeping multiple exposure events at an acceptably low level. The theoretical framework developed here is supported by experimental data. Overall, this study reaffirms the feasibility of the use of flow devices for meaningful laser-induced oxidative labeling studies. At the same time, we provide a theoretical underpinning of this technique that goes beyond previously suggested plug flow models.

Mass Spectrometry Combined with Oxidative Labeling for Exploring Protein Structure and Folding

Lars Konermann et al. — Wed, 04 Aug 2010 22:27:42 PDT

This review discusses various mass spectrometry (MS)-based approaches for exploring structural aspects of proteins in solution. Electrospray ionization (ESI)-MS, in particular, has found fascinating applications in this area. For example, when used in conjunction with solution-phase hydrogen/deuterium exchange (HDX), ESI-MS is a highly sensitive tool for probing conformational dynamics. The main focus of this article is a technique that is complementary to HDX, that is, the covalent labeling of proteins by hydroxyl radicals. The reactivity of individual amino acid side chains with *OH is strongly affected by their degree of solvent exposure. Thus, analysis of the oxidative labeling pattern by peptide mapping and tandem mass spectrometry provides detailed structural information. A convenient method for *OH production is the photolysis of H(2)O(2) by a pulsed UV laser, resulting in oxidative labeling on the microsecond time scale. Selected examples demonstrate the use of this technique for structural studies on membrane proteins, and the combination with rapid mixing devices for characterizing the properties of short-lived protein (un)folding intermediates.

A Paramagnetic Chemical Exchange-based MRI Probe Metabolized by Cathepsin D: Design, Synthesis and Cellular Uptake Studies

Mojmír Suchý et al. — Sun, 20 Jun 2010 18:08:27 PDT

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimer's disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

Membrane Protein Structural Insights from Chemical Labeling and Mass Spectrometry

Yan Pan et al. — Sun, 20 Jun 2010 17:56:57 PDT

Membrane proteins play a central role in virtually all biological processes, and they represent important drug targets. Unfortunately, the application of traditional high-resolution methods such as X-ray crystallography and NMR spectroscopy to membrane proteins remains challenging. This article reviews alternative approaches that involve chemical labeling and mass spectrometry (MS) for gaining insights into membrane protein structure, function, and interactions. Hydrogen/deuterium exchange MS represents an interesting avenue for exploring biomolecular conformations and dynamics, but thus far this technique has not been widely adopted for membrane protein studies. The main focus of this article is on the use of labeling agents that introduce covalent modifications in solvent-accessible protein regions. While it is possible to monitor the occurrence of single-site modifications using traditional biochemical methods or optical spectroscopy, the use of MS greatly enhances the scope and potential of this approach because multiple tagging events can be detected in parallel. The traditional bottom-up workflow of these studies involves the digestion of a chemically labeled membrane protein by a specific protease such as trypsin. This is followed by chromatographic separation of the resulting peptides and on-line electrospray ionization MS. The application of tandem MS allows pinpointing the exact locations of chemical modifications. A particularly exciting aspect is the applicability of covalent labeling techniques to membrane protein within their natural lipid environment, or even inside living cells. Some of these concepts will be illustrated using the oxidative labeling of bacteriorhodopsin as an example, but numerous other labeling agents and protein systems are being highlighted as well. It is hoped that this review will stimulate further developments in the characterization of membrane proteins.

Calcium-Induced Structural Transitions of the Calmodulin−Melittin System Studied by Electrospray Mass Spectrometry: Conformational Subpopulations and Metal-Unsaturated Intermediates

Jingxi Pan et al. — Sat, 08 May 2010 23:39:29 PDT

Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca(4).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM(D) followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.

Synchrotron X-ray Fluorescence and Trace Metals in the Cementum Rings of Human Teeth

Ronald R. Martin et al. — Tue, 09 Mar 2010 00:21:58 PST

Synchrotron micro X-ray fluorescence has been used to study differences in the trace element concentration between the dentine and cementum rings of human teeth. The results show that metals such as zinc are concentrated in the cementum rings. This suggests that if the rings are deposited annually a temporal record of metal exposure may be extracted from this material.

Metal Distributions in the Cementum Rings of Human Teeth: Possible Depositional Chronologies and Diagenesis

Ronald R. Martin et al. — Tue, 09 Mar 2010 00:10:28 PST

Synchrotron radiation analysis has been used to study the trace metal distribution in the cementum rings of selected human teeth. The resulting patterns can be used to identify regions within the root cementum, where the metals are unambiguously endogenous. Since the cementum is thought to display annual growth rings the ability to distinguish endogenous from diagenetic deposition in the trace metal content of the cementum rings, may provide a chronology of an individual's metal exposure.

Comparison of the Distributions of Bromine, Lead, and Zinc in Tooth and Bone from an Ancient Peruvian Burial Site by X-ray Fluorescence

Ronald R. Martin et al. — Tue, 09 Mar 2010 00:02:58 PST

Synchrotron micro X-ray fluorescence was used to study the distribution of selected trace elements (Zn, Pb, and Br) in tooth and bone samples obtained from an individual from a pre-Columbian archaeological site (Cabur) located on the north coast of Peru. The results show that Zn, Pb, and Br are present in both the teeth and bone samples and that the Zn and Pb seem to be confined to similar regions (cementum and periostium), while Br shows a novel distribution with enrichment close to the Haversian canals and (or) in regions that appear to be Ca deficient.

Preliminary Investigation of Element Distribution in the Cementum Rings of Human Teeth Using Time of Flight Secondary Ion Mass Spectroscopy

Ronald R. Martin et al. — Mon, 08 Mar 2010 23:48:19 PST

Time of Flight Secondary Ion Mass Spectroscopy (ToF- SIMS) has been used to study the element distribution in the cementum region of two adult human teeth. The results are consistent with diagenetic changes in the organic components in one of the samples. In addition, lead of uncertain origin is also found in one specimen. In both cases Mg is enriched in the dentine relative to the cementum and there is evidence for leaching of mobile species such as sodium.

A Single-label Phenylpyrrolocytidine Provides a Molecular Beacon-like Response Reporting HIV-1 RT RNase H Activity

Alexander S. Wahba et al. — Sat, 27 Feb 2010 22:08:09 PST

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.

Blue Fluorescent Deoxycytidine Analogues: Convergent Synthesis, Solid-state and Electronic Structure, and Solvatochromism

David W. Dodd et al. — Mon, 08 Feb 2010 16:36:13 PST

We report the synthesis and photospectroscopic characterisation of intrinsically fluorescent triazole-appended cytidines. Fluorescence was found to be highly dependent on solvent conditions. X-Ray crystallographic data show the proton of the exocyclic amine of the nucleobase and the triazole N(3) engaged in a H-bond.

Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

Yan Pan et al. — Sun, 10 Jan 2010 17:11:26 PST

Little is known about the structural properties of semi-denatured membrane proteins. The current study employs laser-induced oxidative labeling of methionine side chains in combination with electrospray mass spectrometry and optical spectroscopy for gaining insights into the conformation of bacteriorhodopsin (BR) under partially denaturing conditions. The native protein shows extensive oxidation at M32, M68, and M163, which are located in solvent-accessible loops. In contrast, M20 (helix A), M56/60 (helix B), M118 (helix D), M145 (helix E), and M209 (helix G) are strongly protected, consistent with the known protein structure. Exposure of the protein to acidic conditions leads to a labeling pattern very similar to that of the native state. The absence of large-scale conformational changes at low pH is in agreement with recent crystallography data. Solubilization of BR in SDS induces loss of the retinal chromophore concomitant with collapse of the binding pocket, thereby precluding solvent access to the protein interior. Tryptophan fluorescence data confirm the presence of a large protein core that remains protected from water. However, oxidative labeling indicates partial unfolding of helices A and D in SDS. Irreversible thermal denaturation of the protein at 100 °C induces a labeling pattern quite similar to that seen upon SDS exposure. Labeling experiments on refolded bacterioopsin reveal a native-like structure, but with partial unfolding of helix D. Our data suggest that noncovalent contacts with the retinal chromophore in native BR play an important role for the stability of this particular helix. Overall, the present work illustrates the viability of using laser-induced oxidative labeling as a novel tool for characterizing structural changes of membrane proteins in response to alterations of their solvent environment.

Cellular Localization and Allele-selective Inhibition of Mutant Huntingtin Protein by Peptide Nucleic Acid Oligomers Containing the Fluorescent Nucleobase [bis-o-(aminoethoxy)phenyl]pyrrolocytosine

Jiaxin Hu et al. — Wed, 06 Jan 2010 10:41:20 PST

Peptide nucleic acid (PNA) is a successful DNA/RNA mimic. A major challenge for research is to invent chemically modified PNAs that retain the favorable properties of the parent compound while improving biological recognition. Here, we test modified PNAs containing [bis-o-(aminoethoxy)phenyl]pyrrolocytosine bases designed to engage guanine with an additional hydrogen bond. We observe elevated melting temperatures, localization to cellular compartments, and allele-selective inhibition of mutant huntingtin protein expression.

Peptide Nucleic Acid Containing a Meta-Substituted Phenylpyrrolocytosine Exhibits a Fluorescence Response and Increased Binding Affinity toward RNA

Filip Wojciechowski et al. — Sun, 03 Jan 2010 15:36:29 PST

Peptide nucleic acids (PNA) containing meta-substituted 6-phenylpyrrolocytosine (PhpC), [mono-m-(aminoethoxy)phenyl]pyrrolocytosine (mmePhpC), [mono-m-(aminopropoxy)phenyl]pyrrolocytosine (mmpPhpC), and [mono-m-(guanidinoethoxy)phenyl]pyrrolocytosine (mmguaPhpC), have been synthesized. Meta-substituted PhpCs have been hybridized with overall higher binding affinity toward DNA and RNA than previously synthesized moePhpC or newly synthesized mopPhpC. The guanidinium-containing nucleobase, mmguaPhpC, exhibited the highest increase in binding affinity toward RNA while fluorometrically responding on the state of hybridization.