20 research outputs found
Structural Characterization of Protein Folding Intermediates by Oxidative Labeling and Mass Spectrometry
A key challenge associated with protein folding studies is the characterization of short-lived intermediates that become populated en route to the native state. In this work, a covalent labeling method was developed that provides insights into the structures of these transient species. Hydroxyl radical (·OH) reacts with solvent-exposed side chains, whereas buried residues are protected. Mass spectrometry is used for monitoring the locations and the extent of labeling. Pulsed ·OH labeling of proteins at selected time points during folding results in high temporal and spatial resolution when compared to existing other labeling methods.
This novel technique was validated by studying the kinetic unfolding and refolding of holomyoglobin (hMb) and cytochrome c (cyt c), respectively. The noncovalent prosthetic heme group in hMb was shown to drastically affect the unfolding pathway. Cyt c refolding was found to fold in a stepwise manner. The population of a misfolded cyt c intermediate was also detected. Results in both cases were in accord with published data.
Many cellular proteins exist as oligomers. Pulsed ·OH labeling method was therefore extended to monitor the folding and assembly of a 22 kDa homodimeric protein, S100A11. Prior to this study very little information regarding the folding mechanism of this protein was available. ·OH labeling reveals that disruption of the native dimer is followed by the formation of non-native hydrophobic contacts within the denatured monomers. The folding/binding pathway was shown to progress through monomeric and dimeric intermediates.
In the final section of this study we applied ·OH labeling to a large monomeric protein that folds to a metastable state. The folding pathway of the 44 kDa protease inhibitor, α1-antitrypsin, was characterized and compared with complementary data from hydrogen/deuterium exchange studies. Our results show that the formation of early tertiary contacts and specific hydrogen bonds guide the protein towards its active, metastable structure. Structural correlation is also seen between a late kinetic species and a previously characterized equilibrium intermediate of a pathogenic mutant.
Overall, the results presented highlight the ability of the technique developed in this work to provide in-depth information about the mechanisms of protein folding
Crowdsourcing hypothesis tests: Making transparent how design choices shape research results
To what extent are research results influenced by subjective decisions that scientists make as they design studies? Fifteen research teams independently designed studies to answer fiveoriginal research questions related to moral judgments, negotiations, and implicit cognition. Participants from two separate large samples (total N > 15,000) were then randomly assigned to complete one version of each study. Effect sizes varied dramatically across different sets of materials designed to test the same hypothesis: materials from different teams renderedstatistically significant effects in opposite directions for four out of five hypotheses, with the narrowest range in estimates being d = -0.37 to +0.26. Meta-analysis and a Bayesian perspective on the results revealed overall support for two hypotheses, and a lack of support for three hypotheses. Overall, practically none of the variability in effect sizes was attributable to the skill of the research team in designing materials, while considerable variability was attributable to the hypothesis being tested. In a forecasting survey, predictions of other scientists were significantly correlated with study results, both across and within hypotheses. Crowdsourced testing of research hypotheses helps reveal the true consistency of empirical support for a scientific claim.</div
Time-Dependent Changes in Side-Chain Solvent Accessibility during Cytochrome c Folding Probed by Pulsed Oxidative Labeling and Mass Spectrometry
The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (.OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as +16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by .OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible
Partially Disordered Proteins Studied by Ion Mobility-Mass Spectrometry: Implications for the Preservation of Solution Phase Structure in the Gas Phase
The
coupling of electrospray ionization (ESI) with ion mobility-mass
spectrometry (IM-MS) allows structural studies on biological macromolecules
in a solvent-free environment. Collision cross sections (CCSs) measured
by IM-MS provide a measure of analyte size. For native proteins and
their complexes, many structural features can be preserved in the
gas phase, making IM-MS a powerful approach for a range of bioanalytical
applications. In addition to tightly folded conformers, a large number
of partially disordered proteins participate in biological processes
and disease mechanisms. It remains unclear to what extent IM-MS is
suitable for exploring structural properties of these semifolded species.
The current work addresses this question, using myoglobin as model
system. This protein follows a sequential unfolding pathway that comprises
two partially disordered states, i.e., apo-myoglobin (aMb) at pH 7
and pH 4. IM-MS data acquired for these two conformers were compared
to those of native holo-myoglobin (hMb) at pH 7 and extensively unfolded
aMb at pH 2. When examining individual aMb charge states, the degree
of gas phase unfolding is not strongly correlated with the corresponding
solution behavior. A key problem is that non-native conformers generate
high ESI charge states, resulting in conformational transitions caused
by intramolecular electrostatic repulsion. It is possible to establish
a link between solution phase and gas phase structure when normalizing
CCS distributions according to their respective ESI-MS signal intensities.
This approach yields CCS averages that follow the expected progression
hMb<sub>pH 7</sub> < aMb<sub>pH 7</sub> < aMb<sub>pH 4</sub> < aMb<sub>pH 2</sub>. However, this trend
mainly reflects the protonation behavior of the conformers during
the ESI process, rather than a genuine memory of solution structure.
Overall, our data reveal that electrostatically driven expansion as
well as collapse events can lead to disparities between gaseous and
solution structures for partially unfolded proteins. IM-MS data on
non-native conformers should therefore be interpreted with caution
Submillisecond Protein Folding Events Monitored by Rapid Mixing and Mass Spectrometry-Based Oxidative Labeling
Kinetic measurements can provide
insights into protein folding
mechanisms. However, the initial (submillisecond) stages of folding
still represent a formidable analytical challenge. A number of ultrarapid
triggering techniques have been available for some time, but coupling
of these techniques with detection methods that are capable of providing
detailed structural information has proven to be difficult. The current
work addresses this issue by combining submillisecond mixing with
laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model
system for our measurements. Exposure of the protein to a brief pulse
of hydroxyl radical (·OH) at different time points during folding
introduces covalent modifications at solvent accessible side chains.
The extent of labeling is monitored using mass spectrometry-based
peptide mapping, providing spatially resolved measurements of changes
in solvent accessibility. The submillisecond mixer used here improves
the time resolution by a factor of 50 compared to earlier ·OH
labeling experiments from our laboratory. Data obtained in this way
indicate that early aMb folding events are driven by both local and
sequence-remote docking of hydrophobic side chains. Assembly of a
partially formed A(E)G(H) scaffold after 0.2 ms is followed by stepwise
consolidation that ultimately yields the native state. Major conformational
changes go to completion within 0.1 s. The technique introduced here
is capable of providing in-depth structural information on very short
time scales that have thus far been dominated by low resolution (global)
spectroscopic probes. By employing submillisecond mixing in conjunction
with slower mixing techniques, it is possible to observe complete
folding pathways, from fractions of a millisecond all the way to minutes
Mapping pH-Induced Protein Structural Changes Under Equilibrium Conditions by Pulsed Oxidative Labeling and Mass Spectrometry
Mass spectrometry (MS)-based protein conformational studies
are
a rapidly growing field. The characterization of partially disordered
conformers is of particular interest because these species are not
amenable to classical high-resolution techniques. Such equilibrium
intermediates can often be populated by exposure to mildly acidic
pH. Hydroxyl radical (·OH) introduces oxidative modifications
at solvent-accessible side chains, while buried sites are protected.
·OH can be generated by laser photolysis of H<sub>2</sub>O<sub>2</sub> (fast photochemical oxidation of proteins–FPOP).
The resulting labeling pattern can be analyzed by MS. The characterization
of partially disordered intermediates usually involves comparative
measurements under different solvent conditions. It can be challenging
to separate structurally induced labeling changes from pH-mediated
“secondary” effects. The issue of secondary effects
in FPOP has received little prior attention. We demonstrate that with
a proper choice of conditions (e.g., in the absence of pH-dependent ·OH
scavengers) such undesired phenomena can be almost completely eliminated.
Using apomyoglobin as a model system, we map the structure of an intermediate
that is formed at pH 4. This species retains a highly protected helix
G that is surrounded by partially protected helices A, B, and H. Our
results demonstrate the utility of FPOP for the structural characterization
of equilibrium intermediates. The near absence of an intrinsic pH
dependence represents an advantage compared to hydrogen/deuterium
exchange MS
Utilizing Microchip Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry
Hydrogen exchange
(HX) mass spectrometry (MS) of complex mixtures
requires a fast, reproducible, and high peak capacity separation prior
to MS detection. The current paradigm relies on liquid chromatography
(LC) with fast gradients performed at low temperatures to minimize
back exchange. Unfortunately, under these conditions, the efficiency
of LC is limited due to resistance to mass transfer, reducing the
capability to analyze complex samples. Capillary electrophoresis (CE),
on the other hand, is not limited by resistance to mass transfer,
enabling very rapid separations that are not adversely affected by
low temperature. Previously, we have demonstrated an integrated microfluidic
device coupling CE with electrospray ionization (ESI) capable of very
rapid and high efficiency separations. In this work, we demonstrate
the utility of this microchip CE-ESI device for HX MS. High speed
CE-ESI of a bovine hemoglobin pepsin digestion was performed in 1
min with a peak capacity of 62 versus a similar LC separation performed
in 7 min with peak capacity of 31. A room temperature CE method performed
in 1.25 min provided similar deuterium retention as an 8.5 min LC
method conducted at 0 °C. Separation of a complex mixture with
CE was done with considerably better speed and nearly triple the peak
capacity than the equivalent separation by LC. Overall, the results
indicate the potential utility of microchip CE-ESI for HX MS
The Resistivity And Thermoelectric-Power Of Liquid Ag-Pd Alloys
The resistivity (?) and the thermoelectric power (S) have been measured as functions of concentration and temperature for the liquid alloy system Ag-Pd. Comparison is made between the experimental results and the theoretical predictions of a nearly free electron model adapted for liquid transition metals. It is concluded that such a theory is unable to reproduce the gross features of the concentration dependence of ? and S even when concentration-dependent effective valencies are introduced. The close similarity between the concentration dependence of the resistivity and the thermoelectric power of solid Ag-Pd alloys at high temperatures and that of the liquid alloys lends support to the alternative s-d scattering model proposed by Mott