182 results on '"Lars Konermann"'
Search Results
152. Chapter 5 Biochemical Reaction Kinetics Studied by Time-Resolved Electrospray Ionization Mass Spectrometry
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Jingxi Pan, Derek J. Wilson, and Lars Konermann
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Chemical kinetics ,Electrospray ,Chemistry ,Capillary action ,Distortion ,Electrospray ionization ,Kinetics ,Analytical chemistry ,Biological system ,Mass spectrometry ,Capillary electrophoresis–mass spectrometry - Abstract
Publisher Summary Time-resolved electrospray ionization-mass spectrometry (ESI-MS) represents a powerful alternative to conventional methods for monitoring the kinetics of rapid biochemical processes. The assembly and use of both static and adjustable capillary mixers for time-resolved ESI-MS are relatively straightforward and usually only require minimal modifications of commercially available ion sources. Care must be taken for the analysis of kinetic data collected under laminar flow conditions because the parabolic velocity profile within the reaction capillary leads to a blurring of the time axis. This distortion can be rectified by considering the specific shape of the age distribution function during data analysis. The highly selective nature of ESI-MS permits multiple coexisting species to be monitored simultaneously. For complex systems, this can result in the acquisition of numerous interdependent intensity-time profiles. Analyzing these data globally increases the accuracy of the extracted numerical parameters, aids the interpretation of the measured kinetics, and facilitates the understanding of reaction mechanisms.
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- 2008
153. Hydrogen/deuterium scrambling during quadrupole time-of-flight MS/MS analysis of a zinc-binding protein domain
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Gilles A. Lajoie, Lars Konermann, Brian H. Shilton, Derek J. Wilson, Brian R. Dempsey, Peter L. Ferguson, and Jingxi Pan
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Ions ,Electrospray ,Protein Folding ,Chemistry ,Protein Conformation ,Protein domain ,Analytical chemistry ,Hydrogen Bonding ,Deuterium ,Dissociation (chemistry) ,Mass Spectrometry ,Analytical Chemistry ,Ion ,Scrambling ,Protein Structure, Tertiary ,Solutions ,Crystallography ,Kinetics ,Isotope Labeling ,Kinetic isotope effect ,Quadrupole ,Gases ,Carrier Proteins ,Hydrogen - Abstract
It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.
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- 2006
154. Exploring the relationship between funneled energy landscapes and two-state protein folding
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Lars Konermann
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Quantitative Biology::Biomolecules ,Protein Folding ,Chemistry ,Protein Conformation ,Entropy ,Conformational entropy ,Models, Theoretical ,Contact order ,Biochemistry ,Random coil ,Folding (chemistry) ,Diffusion ,Crystallography ,Kinetics ,Structural Biology ,Chemical physics ,Lattice protein ,Protein folding ,Folding funnel ,Downhill folding ,Molecular Biology - Abstract
It should take an astronomical time span for unfolded protein chains to find their native state based on an unguided conformational random search. The experimental observation that folding is fast can be rationalized by assuming that protein energy landscapes are sloped towards the native state minimum, such that rapid folding can proceed from virtually any point in conformational space. Folding transitions often exhibit two-state behavior, involving extensively disordered and highly structured conformers as the only two observable kinetic species. This study employs a simple Brownian dynamics model of "protein particles" moving in a spherically symmetrical potential. As expected, the presence of an overall slope towards the native state minimum is an effective means to speed up folding. However, the two-state nature of the transition is eradicated if a significant energetic bias extends too far into the non-native conformational space. The breakdown of two-state cooperativity under these conditions is caused by a continuous conformational drift of the unfolded proteins. Ideal two-state behavior can only be maintained on surfaces exhibiting large regions that are energetically flat, a result that is supported by other recent data in the literature (Kaya and Chan, Proteins: Struct Funct Genet 2003;52:510-523). Rapid two-state folding requires energy landscapes exhibiting the following features: (i) A large region in conformational space that is energetically flat, thus allowing for a significant degree of random sampling, such that unfolded proteins can retain a random coil structure; (ii) a trapping area that is strongly sloped towards the native state minimum.
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- 2006
155. Enzyme conformational dynamics during catalysis and in the 'resting state' monitored by hydrogen/deuterium exchange mass spectrometry
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Lars Konermann and Yu-Hong Liu
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Conformational change ,Spectrometry, Mass, Electrospray Ionization ,Protein Conformation ,Swine ,Kinetics ,Lysozyme ,Biophysics ,Analytical chemistry ,Mass spectrometry ,Arginine ,01 natural sciences ,Biochemistry ,Catalysis ,03 medical and health sciences ,Protein structure ,Structural Biology ,Computational chemistry ,Genetics ,Animals ,Molecular Biology ,030304 developmental biology ,Induced fit ,0303 health sciences ,Chemistry ,010401 analytical chemistry ,Substrate (chemistry) ,Deuterium Exchange Measurement ,Cell Biology ,Deuterium ,Carboxypeptidase B ,0104 chemical sciences ,Steady-state ,Hydrogen–deuterium exchange ,Muramidase ,Steady state (chemistry) ,Chickens - Abstract
This work reports the use of electrospray mass spectrometry for studying the conformational dynamics of enzymes by amide hydrogen/deuterium exchange (HDX) measurements. A rapid-mixing quench-flow approach allows comparisons to be made between the HDX kinetics of free enzymes with those under steady-state conditions. Experiments carried out on carboxypeptidase B in the absence of substrate and in the presence of saturating concentrations of hippuryl-Arg result in HDX kinetics that are indistinguishable. This finding implies that the conformational dynamics that mediate HDX are not significantly different in the resting state of the enzyme and during substrate turnover.
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- 2006
156. A temperature-jump stopped-flow system for monitoring chemical kinetics triggered by rapid cooling
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Lars Konermann and Brian L. Boys
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Chemical kinetics ,Chemistry ,Temperature jump ,Kinetics ,Fluorescence spectrometry ,Biophysics ,Analytical chemistry ,Native state ,Denaturation (biochemistry) ,Protein folding ,Fluorescence spectroscopy ,Analytical Chemistry - Abstract
This work reports the implementation of a stopped-flow system for studying the kinetics of protein folding triggered by rapid cooling. The transition from denaturing temperatures to ambient conditions is achieved by rapid mixing of pre-heated protein solution with cold refolding buffer. The estimated dead-time of the apparatus is 8 ms. Folding kinetics are monitored by fluorescence spectroscopy. Careful tuning of the solution mixing ratio is required for the elimination of baseline artifacts that could mask fluorescence signals originating from protein conformational changes. The viability of the rapid cooling method is demonstrated by applying it to monitor the refolding of thermally denatured cytochrome c. Following a heat exposure time of 3 min, the protein shows multi-exponential folding kinetics, ultimately resulting in a fluorescence level that is virtually indistinguishable from that of the native state. In contrast, incomplete refolding is observed when the heat exposure time is extended to 30 min. This effect may be due to aggregation phenomena affecting the thermally denatured protein. It appears that the rapid cooling approach reported in this work may become a useful tool for temperature-jump studies in various areas of chemistry and biochemistry.
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- 2006
157. Pulsed hydrogen/deuterium exchange MS/MS for studying the relationship between noncovalent protein complexes in solution and in the gas phase after electrospray ionization
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Lars Konermann and Belal M. Hossain
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Chemical ionization ,Spectrometry, Mass, Electrospray Ionization ,Protein mass spectrometry ,Chemistry ,Electrospray ionization ,technology, industry, and agriculture ,Analytical chemistry ,Deuterium Exchange Measurement ,Proteins ,Mass spectrometry ,Tandem mass spectrometry ,Analytical Chemistry ,Solutions ,Hemoglobins ,Tandem Mass Spectrometry ,Multiprotein Complexes ,Mass spectrum ,Hydrogen–deuterium exchange ,Gases ,Protein Structure, Quaternary ,Dimerization - Abstract
Electrospray ionization mass spectrometry (ESI-MS) has become a standard method for monitoring noncovalent protein-protein interactions. Studies employing this approach tend to operate on the premise that the ionic species observed in the mass spectrum directly reflect the corresponding solution-phase protein quaternary structures. However, dissociation or clustering events taking place during ESI may lead to disparities between the ions observed in the mass spectrum and the protein binding state in bulk solution. Recognizing the occurrence of dissociation or clustering artifacts is not straightforward, leading to possible ambiguities in the interpretation of ESI-MS data. This work employs on-line pulsed hydrogen-deuterium exchange (HDX) for probing the origin of various species in the ESI mass spectrum of hemoglobin. In addition to the canonical hemoglobin tetramer, ESI-MS reveals the presence of monomers, dimers, hexamers, and octamers. Tandem mass spectrometry (MS/MS) is used for extracting HDX levels in a subunit-specific manner. Dimeric species exhibit exchange levels that are significantly above those of the tetramer. Monomeric hemoglobin subunits are labeled to an even greater extent. This HDX pattern implies that monomers and dimers do not represent dissociation artifacts generated during ESI. Instead, they are derived from preexisting solution-phase structures. In contrast, hexamers and octamers exhibit HDX levels that resemble those of the tetramer, thus identifying these larger species as nonspecific clustering artifacts. Overall, it appears that the pulsed HDX MS/MS approach introduced in this work represents a widely applicable tool for deciphering the relationship between ESI mass spectra and protein quaternary structures in solution.
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- 2006
158. A capillary mixer with adjustable reaction chamber volume for millisecond time-resolved studies by electrospray mass spectrometry
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Derek J. Wilson and Lars Konermann
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Capillary electrochromatography ,Chemical ionization ,Electrospray ,Chemistry ,Capillary action ,Electrospray ionization ,Mass spectrum ,Analytical chemistry ,Mass spectrometry ,Ion source ,Analytical Chemistry - Abstract
A novel continuous-flow apparatus for on-line kinetic studies of (bio)chemical solution-phase processes by electrospray ionization mass spectrometry (ESI-MS) is described. The device is based on two concentric capillaries. Fluid is released from the inner capillary into the intercapillary space, where it mixes with solution flowing through the outer capillary, thus initiating the reaction of interest. Gas-phase analyte ions are formed near the tip of the outer capillary by pneumatically assisted ESI. This setup allows the mixer to be placed directly within the ion source, thus providing a minimal dead volume of ~8 nL. Time-resolved data can be recorded in both "spectral" and "kinetic" modes. In the former case, the position of the inner capillary is fixed at various points, such that entire mass spectra can be recorded for selected reaction times. For experiments in kinetic mode, the mass spectrometer monitors the signal intensity at selected m/z values, while the inner capillary is continuously pulled back, thus providing intensity-time profiles for specific reactive species. A theoretical framework is developed that allows the measured kinetics to be analyzed by taking into account the effects of laminar flow within the reaction capillary. Failure to take these effects into account results in erroneous rate constants. Studies on the demetalation kinetics of chlorophyll reveal that the apparatus can reliably measure rate constants up to at least 100 s-1. This represents a substantial improvement over previous ESI-MS-based kinetic methods. Spectral mode experiments on the refolding of ubiquitin show the changing proportions of denatured and tightly folded protein subpopulations in solution. When monitored in kinetic mode, the refolding process was found to proceed with a rate constant of 5.2 s-1.
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- 2006
159. Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel laminar flow device
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Lars Konermann and Derek J. Wilson
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Chemical ionization ,Electrospray ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Microchannel ,Chemistry ,Microchemistry ,Cytochromes c ,Laminar flow ,Microfluidic Analytical Techniques ,Mass spectrometry ,Bradykinin ,Analytical Chemistry ,Membrane ,Models, Chemical ,Potassium Permanganate ,Salts ,Semipermeable membrane ,Diffusion (business) - Abstract
The adverse effects of nonvolatile salts on the electrospray (ESI) mass spectra of proteins and other biological analytes are a major obstacle for a wide range of applications. Numerous sample cleanup approaches have been devised to facilitate ESI-MS analyses. Recently developed microdialysis techniques can shorten desalting times down to several minutes, the bottleneck being diffusion of the contaminant through a semipermeable membrane. This work introduces an approach that allows the on-line desalting of macromolecule solutions within tens of milliseconds. The device does not employ a membrane; instead, it uses a two-layered laminar flow geometry that exploits the differential diffusion of macromolecular analytes and low molecular weight contaminants. To maximize desalting efficiency, diffusive exchange between the flow layers is permitted only for such a time as to allow full exchange of salt, while incurring minimal macromolecule exchange. Computer simulations and optical studies show that the device can reduce the salt concentration by roughly 1 order of magnitude, while retaining approximately 70% of the original protein concentration. Application of this approach to the on-line purification of salt-contaminated protein solutions in ESI-MS results in dramatic improvements of both the signal-to-noise ratio and the absolute signal intensity. However, efficient desalting requires the diffusion coefficients of salt and analyte to differ by roughly 1 order of magnitude or more. This technique has potential to facilitate high-throughput analyses of biological macromolecules directly from complex matrixes. In addition, it may become a valuable tool for process monitoring and for on-line kinetic studies on biological systems.
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- 2005
160. Mass spectrometry-based approaches to protein-ligand interactions
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Sonya M. Schermann, Douglas A. Simmons, and Lars Konermann
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Hydrogen exchange ,Chemistry ,Deuterium Exchange Measurement ,Proteins ,Computational biology ,Proteomics ,Mass spectrometry ,Bioinformatics ,Ligands ,Biochemistry ,Mass Spectrometry ,Cellular communication ,Cross-Linking Reagents ,Animals ,Humans ,Molecular Biology ,Protein ligand ,Protein Binding - Abstract
One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein-ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.
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- 2005
161. 2003 Fred Beamish Award Lecture — Exploring the Dynamics of Biological Systems by Mass Spectrometry
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Lars Konermann
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inorganic chemicals ,Chemistry ,Electrospray ionization ,Equilibrium conditions ,Organic Chemistry ,General Chemistry ,General Medicine ,Polypeptide chain ,Mass spectrometry ,Catalysis ,Folding (chemistry) ,Organic chemistry ,Rapid mixing ,Protein folding ,Biological system - Abstract
This review describes the use of electrospray ionization mass spectrometry (ESI-MS) in conjunction with on-line rapid mixing techniques. This combination, termed "time-resolved" ESI-MS, provides a powerful approach for studying solution-phase reactions on timescales as short as a few milliseconds. Of particular interest is the application of this technique for monitoring protein folding reactions. Time-resolved ESI-MS can provide detailed information on structural changes of the polypeptide chain, while at the same time probing the occurrence of noncovalent ligandprotein interactions. Especially when used in combination with hydrogendeuterium pulse labeling, these measurements yield valuable structural information on short-lived folding intermediates. Similar approaches can be used to monitor the dynamics of proteins under equilibrium conditions. Another important application of time-resolved ESI-MS are mechanistic studies on enzyme-catalyzed processes. These reactions can be monitored under presteady-state conditions, without requiring artificial chromophoric substrates or radioactive labeling. We also discuss the use of ESI-MS for monitoring noncovalent ligandprotein interactions by diffusion measurements. In contrast to conventional MS-based techniques, this approach does not rely on the preservation of noncovalent interactions in the gas phase. It appears that diffusion measurements by ESI-MS could become an interesting alternative to existing methods for the high throughput screening of compound libraries in the context of drug discovery.Key words: reaction intermediate, rapid mixing, kinetics, protein conformation, protein function.
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- 2005
162. Kinetic unfolding mechanism of the inducible nitric oxide synthase oxygenase domain determined by time-resolved electrospray mass spectrometry
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Derek J. Wilson, Steven P. Rafferty, and Lars Konermann
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Protein Denaturation ,Protein Folding ,Spectrometry, Mass, Electrospray Ionization ,Time Factors ,Protein Conformation ,Protein subunit ,Dimer ,Analytical chemistry ,Nitric Oxide Synthase Type II ,Biochemistry ,Cofactor ,chemistry.chemical_compound ,Mice ,Protein structure ,Animals ,Denaturation (biochemistry) ,Heme ,biology ,Hydrogen-Ion Concentration ,Protein Structure, Tertiary ,Nitric oxide synthase ,Kinetics ,chemistry ,Models, Chemical ,Heme Oxygenase (Decyclizing) ,biology.protein ,Biophysics ,Oxygenases ,Protein folding ,Nitric Oxide Synthase ,Dimerization - Abstract
The inducible nitric oxide synthase core oxygen domain (iNOS(COD)) is a homodimeric protein complex of ca. 100 kDa. In this work, the subunit disassembly and unfolding of the protein following a pH jump from 7.5 to 2.8 were monitored by on-line rapid mixing in conjunction with electrospray (ESI) time-of-flight mass spectrometry. Various protein species become populated during the denaturation process. These can be distinguished by their ligand binding behavior, and by the different charge states that they produce during ESI. Detailed intensity-time profiles were obtained for all of these species, and the kinetics were subjected to a global analysis which allows a model of the denaturation process to be developed. The data are described well by three relaxation times (tau(1) = 0.36 s, tau(2) = 0.62 s, and tau(3) = 3.3 s), each of which has a characteristic amplitude spectrum. The initial step of the reaction is the disruption of the iNOS(COD) dimer, to generate heme-bound monomeric species in various degrees of unfolding. This first step is accompanied by the loss of two tetrahydrobiopterin cofactors. Subsequent heme loss generates monomeric apoproteins exhibiting various degrees of unfolding. In addition, the formation of proteins that are bound to two heme groups is observed. A subpopulation of holo monomers undergoes substantial unfolding while retaining contact with the heme cofactor. Together with previous studies, the results of this work suggest that the occurrence of complex reaction mechanisms involving several short-lived intermediates is a common feature for the denaturation of large multiprotein complexes.
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- 2005
163. Determination of ligand-protein dissociation constants by electrospray mass spectrometry-based diffusion measurements
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Sonya M. Clark and Lars Konermann
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chemistry.chemical_classification ,Electrospray ,Chemical ionization ,Spectrometry, Mass, Electrospray Ionization ,Time Factors ,Ligand ,Electrospray ionization ,Analytical chemistry ,Mass spectrometry ,Ligands ,Sensitivity and Specificity ,Analytical Chemistry ,Benzamidines ,Dissociation constant ,Diffusion ,Structure-Activity Relationship ,chemistry ,Non-covalent interactions ,Muramidase ,Trypsin ,Diffusion (business) ,Enzyme Inhibitors ,Trisaccharides ,Protein Binding - Abstract
A novel approach for the quantification of ligand-protein interactions is presented. Electrospray ionization mass spectrometry (ESI-MS) is used to monitor the diffusion behavior of noncovalent ligands in the presence of their protein receptors. These data allow the fraction of free ligand in solution to be determined, such that the corresponding dissociation constants can be calculated. A set of conditions is developed that provides an "allowable range" of concentrations for this type of assay. The method is tested by applying it to two different inhibitor-enzyme systems. The dissociation constants measured for benzamidine-trypsin and for N,N',N' '-triacetylchitotriose-lysozyme are (50 +/- 10) and (6 +/- 1) mM, respectively. Both of these results are in good agreement with previous data from the literature. In contrast to traditional ESI-MS-based methods, the approach used in this work does not rely on the preservation of specific solution-type noncovalent interactions in the gas phase. It is shown that this method allows an accurate determination of dissociation constants, even in cases in which the ion abundance ratio of free to ligand-bound protein in ESI-MS does not reflect the corresponding concentration ratio in solution.
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- 2004
164. Subunit disassembly and unfolding kinetics of hemoglobin studied by time-resolved electrospray mass spectrometry
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Lars Konermann, Gilles A. Lajoie, Douglas A. Simmons, and Amanda Doherty-Kirby, and Derek J. Wilson
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Protein Denaturation ,Protein Folding ,Spectrometry, Mass, Electrospray Ionization ,Electrospray ionization ,Dimer ,Population ,Mass spectrometry ,Biochemistry ,chemistry.chemical_compound ,Hemoglobins ,Tetramer ,Animals ,Denaturation (biochemistry) ,education ,Protein Structure, Quaternary ,Methemoglobin ,Acetic Acid ,education.field_of_study ,Chemistry ,Globins ,Crystallography ,Matrix-assisted laser desorption/ionization ,Kinetics ,Protein Subunits ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mass spectrum ,Cattle ,Apoproteins ,Dimerization - Abstract
We report the use of electrospray ionization (ESI) mass spectrometry (MS) in conjunction with online rapid mixing to monitor the kinetics of acid-induced ferrihemoglobin denaturation. Under equilibrium conditions, the hemoglobin mass spectrum is dominated by the intact heterotetramer. Dimeric and monomeric species are also observed at lower intensities. In addition, ionic signals corresponding to hexameric (tetramer-dimer) and octameric (tetramer x 2) hemoglobin species are observed. These complexes may represent weak solution-phase assemblies. The acid-induced denaturation process was monitored for reaction time ranging from 9 ms to approximately 3 s. The data obtained were subjected to a global analysis procedure which simultaneously fit all kinetic (ESI-MS intensity vs time) profiles to multiexponential expressions. Results of the global analysis are consistent with the coexistence of two subpopulations of tetrameric hemoglobin which differ in their disassembly rates and ESI charge states. The higher-charge state tetramer ions preferentially dissociate via a rapid pathway (tau(1) = 51 ms), resulting in the transient formation of a heme-saturated dimer, holo-alpha-globin, and a heme-deficient dimer. The latter is shown by MS/MS to be comprised of a heme-bound alpha-subunit complexed with an apo-beta-chain. The slow-decaying tetramer population, apparent at a slightly lower average charge state, breaks down into its monomeric constituents with no observable intermediate species (tau(2) = 390 ms). Surprisingly, unfolded apo-alpha-globin is formed more rapidly than unfolded apo-beta-globin. The appearance of the latter occurs with a relaxation time tau(3) of 1.2 s. It is postulated that accumulation of unfolded apo-beta-globin is delayed by transient population of an undetected unfolding intermediate.
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- 2004
165. Effects of ground loop currents on signal intensities in electrospray mass spectrometry
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Lars Konermann and Richard A. Ochran
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Electrospray ,Spectrometry, Mass, Electrospray Ionization ,Reserpine ,Chemistry ,Metallocenes ,Electrospray ionization ,010401 analytical chemistry ,Analytical chemistry ,Electrolyte ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,Ion source ,0104 chemical sciences ,Ion ,Choline ,Structural Biology ,Ionization ,Electrochemistry ,Ground loop (electricity) ,Ferrous Compounds ,Oxidation-Reduction ,Spectroscopy - Abstract
The occurrence of electrochemical processes during the operation of an electrospray ionization (ESI) source is well established. In the positive ion mode, electrons are drawn from the ESI metal capillary to a high voltage power supply. These electrons are the product of charge-balancing oxidation reactions taking place at the liquid/metal interface of the ion source. In a recent study, (Anal. Chem.2001, 73, 4836-4844), our group has shown that the introduction of a ground loop can dramatically enhance the rate of these oxidation processes. Such a ground loop can be introduced by connecting the sample infusion syringe (or the liquid chromatography column, in the case of LC-MS studies) to ground. The magnitude of the ground loop current can be controlled by the electrolyte concentration in the analyte solution, and by the dimensions of the capillary connecting the syringe needle and the ESI source. Using ferrocene as a model system, it is demonstrated that the introduction of such a ground loop can significantly enhance the signal intensity of analytes that form electrochemically ionized species during ESI. However, analytes that form protonated molecular ions, such as reserpine, also show higher signal intensities when a ground loop is introduced into the system. This latter observation is attributed to the occurrence of electrolytic solvent (acetonitrile and/or water) oxidation processes. These reactions generate protons within the ion source, and thus facilitate the formation of [M + nH](n+) ions. Overall, this work provides an example of how the careful control of electrochemical parameters can be exploited to optimize signal intensities in ESI-MS.
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- 2004
166. Mechanistic studies on enzymatic reactions by electrospray ionization MS using a capillary mixer with adjustable reaction chamber volume for time-resolved measurements
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Derek J. Wilson and Lars Konermann
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Chemical ionization ,Electrospray ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Time Factors ,Chemistry ,Electrospray ionization ,Hydrolysis ,Kinetics ,Analytical chemistry ,Acetylation ,Mass spectrometry ,Bradykinin ,Michaelis–Menten kinetics ,Analytical Chemistry ,Nitrophenols ,Reaction rate constant ,Mass spectrum ,Chymotrypsin - Abstract
Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a "makeup solvent" immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.
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- 2004
167. Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions
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Lars Konermann and Sonya M. Clark
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chemistry.chemical_classification ,Protein Denaturation ,Spectrometry, Mass, Electrospray Ionization ,Aqueous solution ,Acetonitriles ,Myoglobin ,Electrospray ionization ,Fluorescence spectrometry ,Analytical chemistry ,Fluorescence correlation spectroscopy ,Heme ,Salt bridge (protein and supramolecular) ,Diffusion ,Solutions ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Non-covalent interactions ,Animals ,Horses ,Dispersion (chemistry) ,Muscle, Skeletal ,Spectroscopy ,Protein Binding - Abstract
This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002, 16, 1454–1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, “harsh” conditions at the ion source are required to disrupt any potential gas- phase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme–protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation.
- Published
- 2003
168. Pre-Steady-State Kinetics of Enzymatic Reactions Studied by Electrospray Mass Spectrometry with On-Line Rapid-Mixing Techniques
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Donald J. Douglas and Lars Konermann
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Electrospray ,chemistry.chemical_compound ,Chromatography ,Resolution (mass spectrometry) ,chemistry ,Formic acid ,Electrospray ionization ,Substrate (chemistry) ,Mass spectrometry ,Enzyme catalysis ,Adduct - Abstract
This chapter focuses on recent developments that allow the direct on-line monitoring of enzymecatalyzed reactions by electrospray ionization (ESI) mass spectrometry (MS). In these experiments, an enzyme/substrate mixture is injected directly into the ESI source of the mass spectrometer, while the reaction proceeds in solution. With this approach it is possible to overcome many limitations of traditional kinetic methods. The new ESI MS-based techniques have the potential to become standard tools for studying enzymatic reactions in the pre-steady-state regime. These techniques are useful for kinetic studies in other fields of chemistry and biochemistry. The current time resolution of these techniques (tens of milliseconds) is not as good as that of conventional quench-flow and stopped-flow experiments. Certain restrictions may be encountered when choosing solvent systems that are compatible with on-line monitoring of the reaction mixture by ESI MS. Salts, detergents, and pH buffers may interfere with the electrospray process, leading to low signal intensities and the formation of adduct species. The electrospray process is compatible with pH buffers that are based on volatile components, such as acetic acid, formic acid, ammonia, or piperidine. It might be feasible to develop microdialysis systems for kinetic experiments that allow the rapid desalting of the reaction mixture immediately before it is analyzed by ESI MS.
- Published
- 2002
169. Contributors to Volume 354
- Author
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R. Donald Allison, Adolfo Amici, Marcus D. Ballinger, David Barford, Alcira Batlle, Didier Blanot, Susan K. Boehlein, Ahmed Bouhss, Bruce P. Branchaud, Michael Bruner, Jared L. Cartwright, Christopher H. Chang, Timothy D.H. Bugg, Hung-Wei Chih, Jean-franÇois Collet, Paul F. Cook, SÉbastien Dementin, Dominique Deville-Bonne, M.L. Dodson, Donald J. Douglas, H. Brian Dunford, Dale E. Edmondson, Monica Emanuelli, Sarah M. Fleming, Perry A. Frey, Sandaruwan Geeganage, Astrid GrÄs Lund, Jeffrey W. Gross, Sanghwsa Han, Jun Hiratake, Benjamin A. Horenstein, Marja S. Huhta, Boi Hanh Huynh, Makoto Inoue, JoËl Janin, William E. Karsten, Lars Konermann, Carsten Krebs, Andrew J. Kurtz, G. John Langley, Paul A. Lindahl, R. Stephen Lloyd, Prashanti Madhavapeddi, Giulio Magni, Leah A. Marquez-curtis, E. Neil G. Marsh, Alexander G. Mclennan, Henry M. Miziorko, Renee M. Mosi, Claudine Parquet, Daniel L. Purich, Nadia Raffaelli, George H. Reed, Nigel G.J. Richards, Thomas A. Robertson, David R. Rose, Denis L. Rousseau, IpsIta Roymoulik, Silverio Ruggieri, Kanzo Sakata, Gunter Schneider, Holly G. Schnizer, Sheldon M. Schuster, Georg A. Sprenger, Jon D. Stewart, Vincent Stroobant, Dennis J. Stuehr, Peter A. Tipton, B. Elizabeth Turner, Jean Van Heijenoort, Emile Van Schaftingen, Dmitriy A. Vinarov, Zhi-qiang Wang, Chin-chuan Wei, Jacqueline Wicki, and Stephen G. Withers
- Subjects
Volume (thermodynamics) ,Petroleum engineering ,Environmental science - Published
- 2002
170. Protein Folding and Binding Characterized by Mass Spectrometry
- Author
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Modupeola A. Sowole, Siavash Vahidi, and Lars Konermann
- Subjects
Solvent ,Folding (chemistry) ,chemistry.chemical_compound ,Crystallography ,Chemistry ,Covalent bond ,Biophysics ,Side chain ,Protein folding ,Hydroxyl radical ,Hydrogen–deuterium exchange ,Mass spectrometry - Abstract
This presentation will illustrate how the combination of solution phase labeling with mass spectrometry (MS) can elucidate mechanistic aspects of protein behavior. We will focus on two ongoing projects in our laboratory. (A) The initial (submillisecond) stages of protein folding represent a formidable experimental challenge. We have begun to address this issue by using submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for these 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 MS-based peptide mapping, providing spatially-resolved measurements of changes in solvent accessibility. The technique introduced here is capable of providing in-depth structural information on time scales that have thus far been dominated by low resolution spectroscopic probes. (B) The bacterial protease ClpP is a multi-subunit complex with a central degradation chamber that can be accessed via axial pores. In free ClpP these pores are obstructed. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind ClpP and cause the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. We use hydrogen/deuterium exchange MS to obtain insights into the ClpP behavior with and without ADEP1. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore.
- Published
- 2014
171. From small-molecule reactions to protein folding: studying biochemical kinetics by stopped-flow electrospray mass spectrometry
- Author
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Beata M. Kolakowski and Lars Konermann
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Chlorophyll ,Protein Denaturation ,Protein Folding ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Time Factors ,Myoglobin ,Electrospray ionization ,Chlorophyll A ,Kinetics ,Selected reaction monitoring ,Biophysics ,Analytical chemistry ,Cell Biology ,Reaction intermediate ,Mass spectrometry ,Biochemistry ,Chemical kinetics ,chemistry.chemical_compound ,Reaction rate constant ,chemistry ,Molecular Biology - Abstract
This work introduces stopped-flow electrospray ionization (ESI) mass spectrometry (MS) as a method for studying fast biochemical reaction kinetics. After initiating a reaction by rapid mixing of two solutions, the mixture is transferred to a reaction vessel and a steady liquid flow to the ESI source of the mass spectrometer is established. The kinetics are studied in real time by monitoring selected ion intensities as a function of time. In order to characterize the performance of this setup the acid-induced demetallation of chlorophyll a was studied. It was found that the reaction is second order in acid concentration and that pseudo-first-order rate constants of up to roughly 7 s−1 can be measured reliably. Stopped-flow ESI MS was also applied to study the acid-induced denaturation of myoglobin. The data presented here confirm the occurrence of a short-lived unfolding intermediate during this reaction. Stopped-flow ESI MS can provide information that is not accessible by optical rapid-mixing experiments. Therefore it appears that this novel technique has the potential to become a standard tool for kinetic studies in a number of different fields.
- Published
- 2001
172. Reconstitution of acid-denatured holomyoglobin studied by time-resolved electrospray ionization mass spectrometry
- Author
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Vincent W. S. Lee, Yu-Luan Chen, and Lars Konermann
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Electrospray ,Protein Denaturation ,Protein Folding ,Hemeprotein ,Chromatography ,Chemistry ,Myoglobin ,Electrospray ionization ,Heme ,Hydrogen-Ion Concentration ,Mass spectrometry ,Mass Spectrometry ,Analytical Chemistry ,Kinetics ,Protein structure ,Denaturation (biochemistry) ,Protein folding ,Protein ligand ,Acetic Acid - Abstract
Time-resolved electrospray ionization (ESI) mass spectrometry (MS) is a new technique for studying the kinetics of protein folding reactions. It can monitor both changes in the protein conformation and the loss or binding of protein ligands as a function of time. Time-resolved ESI MS was previously used to monitor the acid-induced unfolding of holomyoglobin (hMb). The native form of this protein is characterized by a tightly folded conformation and a heme group that is noncovalently attached to the protein. Acid-induced denaturation induces substantial unfolding of the polypeptide chain and disruption of the heme-protein interactions. In this work, time-resolved ESI MS is used to study the reverse reaction, i.e., reconstitution of acid-denatured hMb. To examine the mechanism and the kinetics of this reaction, a continuous-flow setup with two sequential mixing steps was developed. The data presented in this work show that reconstitution involves the formation of various short-lived intermediates such as tightly folded myoglobin without a heme group and several nativelike forms of the protein that are bound to more than one heme. The occurrence of these transient states is most likely due to the rapid aggregation of free heme in solution.
- Published
- 1999
173. Electron Capture Dissociation of Electrosprayed Protein Ions for Spatially Resolved Hydrogen Exchange Measurements
- Author
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Jingxi Pan, Jun Han, Christoph H. Borchers, and Lars Konermann
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Spectrometry, Mass, Electrospray Ionization ,Hydrogen ,Electron-capture dissociation ,Ubiquitin ,Analytical chemistry ,Deuterium Exchange Measurement ,chemistry.chemical_element ,General Chemistry ,Deuterium ,Mass spectrometry ,Biochemistry ,Catalysis ,Scrambling ,Ion ,Colloid and Surface Chemistry ,Fragmentation (mass spectrometry) ,chemistry ,Chemical physics ,Intramolecular force - Abstract
Mass spectrometry (MS) methods involving gas-phase fragmentation hold considerable promise for analyzing regioselective deuteration patterns of proteins following solution-phase amide hydrogen exchange (HX). However, the general viability of such an approach is questionable due to the possible occurrence of intramolecular hydrogen migration ("scrambling"), which tends to randomize or distort the spatial isotope distribution. Rand et al. (J. Am. Chem. Soc. 2008, 130, 1341-1349) have recently reported the application of electron capture dissociation (ECD) for measuring deuteration patterns of short peptides with very little scrambling by FT-MS. The current work shows that even much larger systems such as the 76-residue protein ubiquitin can be successfully analyzed by ECD following solution-phase HX. The resulting c and z. ion deuteration levels are in remarkable agreement with previous NMR data, demonstrating that the extent of scrambling and/or other gas-phase artifacts is negligible. These results open the door to future experiments on the folding, structure, and dynamics of proteins by HX/ECD-FT-MS.
- Published
- 2008
174. Acid-induced denaturation of myoglobin studied by time-resolved electrospray ionization mass spectrometry
- Author
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Mauk Ag, Donald J. Douglas, Lars Konermann, and Federico I. Rosell
- Subjects
Protein Denaturation ,Protein Folding ,Heme binding ,Absorption spectroscopy ,Myoglobin ,Electrospray ionization ,Analytical chemistry ,Heme ,Chromophore ,Hydrogen-Ion Concentration ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Kinetics ,chemistry ,Mass spectrum ,Animals ,Denaturation (biochemistry) ,Acetic Acid - Abstract
The acid-induced denaturation of holo-myoglobin (hMb) following a pH-jump from 6.5 to 3.2 has been studied by electrospray ionization (ESI) mass spectrometry in combination with a continuous flow mixing technique (time-resolved ESI MS). Different protein conformations are detected by the different charge state distributions that they generate during ESI. The changes in intensity of the peaks in the mass spectrum as a function of time can be described by two exponential lifetimes of 0.38 +/- 0.06 s and 6.1 +/- 0.5 s, respectively. The acid-induced denaturation of hMb was also studied in stopped-flow experiments by monitoring changes in the Soret absorption. The lifetimes measured by this method are in good agreement with those obtained by time-resolved ESI MS. The shorter lifetime is associated with the formation of a transient intermediate which shows the mass of the intact heme-protein complex but leads to the formation of much higher charge states during ESI than native hMb at pH 6.5. This form of hMb has an absorption spectrum similar to that of the native protein, indicating a relatively unperturbed chromophore environment inside the heme binding pocket. The intermediate can thus be characterized as an unfolded form of hMb with essentially intact heme-protein interactions. The longer of the two lifetimes is associated with the formation of a product which has a blue-shifted absorption spectrum with a much lower maximum absorption coefficient than observed for native hMb. In the ESI mass spectrum, this product appears as the apoprotein with high charge states which indicates the disruption of the native heme-protein interactions and a considerable degree of unfolding compared to native apo-myoglobin. The mechanism of acid-induced denaturation of hMb, therefore, appears to follow the sequence (heme-protein)native --> (heme-protein)unfolded --> heme + (protein)unfolded.
- Published
- 1997
175. A long lifetime component in the tryptophan fluorescence of some proteins
- Author
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Klaus Döring, Lars Konermann, Fritz Jähnig, and Thomas Surrey
- Subjects
Lactose permease ,Time Factors ,Monosaccharide Transport Proteins ,Protein Conformation ,Molecular Sequence Data ,Biophysics ,Context (language use) ,Peptide ,Biophysical Phenomena ,Fluorescence ,Azurin ,Animals ,Amino Acid Sequence ,Ribonuclease T1 ,Protein secondary structure ,Serum Albumin ,chemistry.chemical_classification ,Symporters ,Chemistry ,Escherichia coli Proteins ,Tryptophan ,Membrane Transport Proteins ,Proteins ,General Medicine ,Rats ,Crystallography ,Membrane ,Spectrometry, Fluorescence ,Membrane protein ,Peptides ,Bacterial Outer Membrane Proteins - Abstract
The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T1, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T1, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypeptide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.
- Published
- 1995
176. Modelling the Low Temperature Fluorescence Kinetics of the Photosystem II Reaction Center: Energy Transfer and Radical Pair Relaxation
- Author
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Guido Gatzen, Alfred R. Holzwarth, and Lars Konermann
- Subjects
Photosynthetic reaction centre ,P700 ,Photosystem II ,Chemistry ,Fluorescence kinetics ,Energy transfer ,Relaxation (physics) ,Photochemistry - Published
- 1995
177. Conformational Dynamics of Partially Denatured Myoglobin Studied by Time-Resolved Electrospray Mass Spectrometry with Online Hydrogen−Deuterium Exchange
- Author
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Douglas A. Simmons, Stanley D. Dunn, and Lars Konermann
- Subjects
Biochemistry - Published
- 2003
178. Molecular Dynamics Simulations of Electrosprayed Water Nanodroplets: Internal Potential Gradients, Location of Excess Charge Centers, and “Hopping” Protons.
- Author
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Elias Ahadi and Lars Konermann
- Subjects
- *
MOLECULAR dynamics , *SIMULATION methods & models , *ELECTROSPRAY ionization mass spectrometry , *PROTON transfer reactions , *COULOMB potential , *SODIUM ions , *ELECTROSTATICS , *HOPPING conduction - Abstract
Water nanodroplets charged with excess protons play a central role during electrospray ionization (ESI). In the current study molecular dynamics (MD) simulations were used for gaining insights into the nanodroplet behavior based on classical mechanics. The SPC/E water model was modified to permit the inclusion of protons as highly mobile point charges at minimum computational cost. A spherical trapping potential was assigned to every SPC/E oxygen, thereby allowing the formation of protonated water molecules. Within a tightly packed nanodroplet the individual potential wells merge to form a three-dimensional energy landscape that facilitates rapid proton hopping between water molecules. This approach requires short-range modifications to the standard Coulomb potential for modeling electrostatic proton−water interactions. Simulations on nanodroplets consisting of 1248 water molecules and 10 protons (radius, ca. 21 Å) result in a proton diffusion coefficient that is in agreement with the value measured in bulk solution. Radial proton distributions extracted from 1 ns MD runs exhibit a large peak around 14 Å, in addition to substantial population density closer to the droplet center. Similar radial distributions were found for nanodroplets charged with Na+ions. This behavior is dramatically different from that expected on the basis of continuum electrostatic theory, which predicts that excess charge should be confined to a thin layer on the droplet surface. One important contributor to this effect seems to be the ordering of water molecules at the liquid/vacuum interface. This ordering results in an electrical double layer, generating a potential gradient that tends to pull positive charge carriers (such as protons, but also others such as Na+ions) toward the droplet interior. This deviation from the widely assumed surface charge paradigm could have implications for the mechanism by which protonated analyte ions are formed during ESI. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
179. A Minimalist Model for Exploring Conformational Effects on the Electrospray Charge State Distribution of Proteins.
- Author
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Lars Konermann
- Subjects
- *
PROTEIN analysis , *CONFORMATIONAL analysis , *ELECTROSTATICS , *ELECTROSPRAY ionization mass spectrometry - Abstract
The electrospray ionization (ESI) charge state distribution of proteins is highly sensitive to the protein structure in solution. Unfolded conformations generally form higher charge states than tightly folded structures. The current study employs a minimalist molecular dynamics model for simulating the final stages of the ESI process in order to gain insights into the physical reasons underlying this empirical relationship. The protein is described as a string of 27 beads (“residues”), 9 of which are negatively charged and represent possible protonation sites. The unfolded state of this bead string is a random coil, whereas the native conformation adopts a compact fold. The ESI process is simulated by placing the protein inside a solvent droplet with a 2.5 nm radius consisting of 1600 Lennard-Jones particles. In addition, the droplet contains 14 protons which are modeled as highly mobile point charges. Disintegration of the droplet rapidly releases the protein into the gas phase, resulting in average charge states of 4.8 and 7.4 for the folded and unfolded conformation, respectively. The protonation probabilities of individual residues in the folded state reveal a characteristic pattern, with values ranging from 0.2 to 0.8. In contrast, the protonation probabilities of the unfolded protein are more uniform and cover the range from 0.8 to 1.0. The origin of these differences can be traced back to a combination of steric and electrostatic effects. Residues exhibiting a small accessible surface area are less likely to capture a proton, an effect that is exacerbated by partial electrostatic shielding from nearby positive residues. Conversely, sites that are sterically exposed are associated with electrostatic funnels that greatly increase the likelihood of protonation. Unfolding enhances the steric and electrostatic exposure of protonation sites, thereby causing the protein to capture a greater number of protons during the droplet disintegration process. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
180. Correction of Both NBD1 Energetics and Domain Interface Is Required to Restore ΔF508 CFTR Folding and Function
- Author
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Gergely L. Lukacs, Salvatore di Bernardo, Kai Du, Wael M. Rabeh, Florian Bossard, Ariel Roldan, Haijin Xu, Miklós Bagdány, Yu-Hong Liu, Cory M. Mulvihill, Tsukasa Okiyoneda, and Lars Konermann
- Subjects
Models, Molecular ,Protein Folding ,Cystic Fibrosis Transmembrane Conductance Regulator ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Humans ,ΔF508 ,030304 developmental biology ,0303 health sciences ,Biochemistry, Genetics and Molecular Biology(all) ,Endoplasmic reticulum ,Cystic fibrosis transmembrane conductance regulator ,Protein Structure, Tertiary ,Folding (chemistry) ,Biochemistry ,Membrane protein ,Mutation ,Biophysics ,biology.protein ,Protein folding ,030217 neurology & neurosurgery ,Biogenesis - Abstract
SummaryThe folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508 CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.
- Full Text
- View/download PDF
181. A Simple Model for the Disintegration of Highly Charged Solvent Droplets during Electrospray Ionization
- Author
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Lars Konermann
- Subjects
Work (thermodynamics) ,Electrospray ,Fission ,Chemistry ,Analytical chemistry ,Charge (physics) ,Medical Biochemistry ,Charged particle ,eye diseases ,Stress (mechanics) ,Protein filament ,Surface tension ,Physics::Fluid Dynamics ,Structural Biology ,Chemical physics ,Spectroscopy - Abstract
This work uses a minimalist model for deciphering the opposing effects of Coulomb repulsion and surface tension on the stability of electrosprayed droplets. Guided by previous observations, it is assumed that progeny droplets are ejected from the tip of liquid filaments that are formed as protrusions of an initially spherical parent. Nonspherical shapes are approximated as assemblies of multiple closely spaced beads. This strategy greatly facilitates the calculation of electrostatic and surface energies. For a droplet at the Rayleigh limit the model predicts that growth of a very thin filament is a spontaneous process with a negligible activation barrier. In contrast, significant barriers are encountered for the formation of larger diameter filaments. These different barrier heights favor highly asymmetric droplet fission because the dimensions of the filament determine those of the ejected droplet(s). Substantial charge accumulation occurs at the filament termini. This allows each progeny droplet to carry a significant fraction of charge, despite its very small volume. In the absence of a long connecting filament, relieving electrostatic stress through progeny droplet emission would be ineffective. The model predicts the prevalence of fission events leading to the formation of several progeny droplets, instead of just a single one. Ejection bursts are followed by collapse back to a spherical shape. The resulting charge depleted system is incapable of producing additional progeny droplets until solvent evaporation returns it to the Rayleigh limit. Despite the very simple nature of the model used here, all of these predictions agree with experimental data.
- Full Text
- View/download PDF
182. Analysis of Protein Mixtures by Electrospray Mass Spectrometry: Effects of Conformation and Desolvation Behavior on the Signal Intensities of Hemoglobin Subunits
- Author
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Brian L. Boys, Lars Konermann, and Mark C. Kuprowski
- Subjects
Electrospray ,Spectrometry, Mass, Electrospray Ionization ,Hemeprotein ,Chemistry ,Protein Conformation ,Electrospray ionization ,Protein subunit ,010401 analytical chemistry ,Analytical chemistry ,Complex Mixtures ,Hydrogen-Ion Concentration ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,0104 chemical sciences ,Ion ,Solvent ,Hemoglobins ,Protein Subunits ,Structural Biology ,Solvents ,Hemoglobin ,Spectroscopy - Abstract
The determination of solution-phase protein concentration ratios based on ESI-MS intensity ratios is not always straightforward. For example, equimolar mixtures of hemoglobin alpha- and beta-subunits consistently result in much higher peak intensities for the alpha-chain. The current work explores the origin of this effect. Under mildly acidic conditions (pH 3.4) alpha-globin is extensively unfolded, whereas beta-globin retains residual structure. Because of its greater nonpolar character, the more unfolded alpha-subunit can more effectively compete for charge. This leads to suppression of beta-globin signals under conditions where the protein ion yield is limited by the charge concentration on the initially formed ESI droplets. More balanced intensities are observed when operating under charge excess conditions and/or in a solvent environment where both proteins are unfolded to a similar degree (pH 2.2). However, even in these cases the overall alpha-globin peak intensity is still twice as high as that of the beta-subunit. The persistent imbalance under these conditions originates from the different declustering behaviors of the two proteins. A considerable fraction of beta-globin undergoes incomplete desolvation during ESI, thereby reducing the intensity of bare [beta + zH](z+) ions. When including the contributions of incompletely desolvated species, the overall alpha:beta ion intensity ratio is close to unity. The alpha:beta intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) dissimilarities in the protein desolvation behavior.
- Full Text
- View/download PDF
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