Patrik Lundström - Academia.edu (original) (raw)

Papers by Patrik Lundström

Research paper thumbnail of Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE

Proceedings of the National Academy of Sciences, 2010

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cyto... more MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae , with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for fu...

Research paper thumbnail of Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain

PLOS ONE, 2017

Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-... more Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-box and coiled-coil domains (RBCC), where the B-box domain is a unique feature of the TRIM protein family. TRIM21 is an E3 ubiquitin-protein ligase implicated in innate immune signaling by acting as an autoantigen and by modifying interferon regulatory factors. Here we report the three-dimensional solution structure of the TRIM21 B-box2 domain by nuclear magnetic resonance (NMR) spectroscopy. The structure of the B-box2 domain, comprising TRIM21 residues 86-130, consists of a short α-helical segment with an N-terminal short β-strand and two anti-parallel β-strands jointly found the core, and adopts a RINGlike fold. This ββαβ core largely defines the overall fold of the TRIM21 B-box2 and the coordination of one Zn 2+ ion stabilizes the tertiary structure of the protein. Using NMR titration experiments, we have identified an exposed interaction surface, a novel interaction patch where the B-box2 is likely to bind the N-terminal RING domain. Our structure together with comparisons with other TRIM B-box domains jointly reveal how its different surfaces are employed for various modular interactions, and provides extended understanding of how this domain relates to flanking domains in TRIM proteins.

Research paper thumbnail of Measurement of protein backbone 13CO and 15N relaxation dispersion at high resolution

Journal of Biomolecular NMR, 2017

Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics ... more Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates Maxim Mayzel and Alexandra Ahlner have contributed equally to this work.

Research paper thumbnail of Structural insights into the calcium-mediated allosteric transition in the C-terminal domain of calmodulin from NMR measurements

Biochemistry, Jan 30, 2015

Calmodulin is a two-domain signalling protein that becomes activated upon binding cooperatively t... more Calmodulin is a two-domain signalling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin function, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and NMR experiments on the Ca2+-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states, and validated such ensemble against three independent experimental datasets, namely inter-proton NOEs, 15N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the correspo...

Research paper thumbnail of Robust and convenient analysis of protein thermal and chemical stability

Protein Science, 2015

We present the software CDpal that is used to analyze thermal and chemical denaturation data to o... more We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

Research paper thumbnail of Local destabilization of the metal-binding region in human copper-zinc superoxide dismutase by remote mutations is a possible determinant for progression of ALS

Biochemistry, Jan 20, 2015

More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismut... more More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to ...

Research paper thumbnail of Isotope Labeling Methods for Relaxation Measurements

Advances in Experimental Medicine and Biology, 2012

Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynami... more Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly (15)N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on (13)Cα, (13)Cβ or (13)CO or (1)Hα. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter.

Research paper thumbnail of Isotope Labeling Methods for Large Systems

Advances in Experimental Medicine and Biology, 2012

A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is th... more A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is that the technique has been limited to relatively small molecules. However, in the last two decades the size limit has been pushed upwards considerably and it is now possible to use NMR spectroscopy for structure calculations of proteins of molecular weights approaching 100 kDa and to probe dynamics for supramolecular complexes of molecular weights in excess of 500 kDa. Instrumental for this progress has been development in instrumentation and pulse sequence design but also improved isotopic labeling schemes that lead to increased sensitivity as well as improved spectral resolution and simplification. These are described and discussed in this chapter, focusing on labeling schemes for amide proton and methyl proton detected experiments. We also discuss labeling methods for other potentially useful positions in proteins.

Research paper thumbnail of Protein Conformational Dynamics by Relaxation Dispersion

Encyclopedia of Biophysics, 2013

Research paper thumbnail of Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches

PLoS computational biology, 2015

The process of resonance assignment is fundamental to most NMR studies of protein structure and d... more The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software...

Research paper thumbnail of Measuring the Signs of 1Hα Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R1ρ NMR Spectroscopy

Journal of the American Chemical Society, 2009

Research paper thumbnail of PINT: a software for integration of peak volumes and extraction of relaxation rates

Journal of Biomolecular NMR, 2013

We present the software PINT (Peak INTegration), designed to perform integration of peaks in NMR ... more We present the software PINT (Peak INTegration), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

Research paper thumbnail of Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Journal of Biomolecular NMR, 2008

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectrosc... more Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

Research paper thumbnail of Quantifying millisecond exchange dynamics in proteins by CPMG relaxation dispersion NMR using side-chain 1H probes

Journal of the American Chemical Society, Jan 15, 2012

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millise... more A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstra...

Research paper thumbnail of Microsecond Protein Dynamics Measured by 13Cα Rotating-Frame Spin Relaxation

ChemBioChem, 2005

NMR spin relaxation in the rotating frame (R1rho) is a unique method for atomic-resolution charac... more NMR spin relaxation in the rotating frame (R1rho) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond timescale. We present a rotating-frame 13C(alpha) relaxation dispersion experiment for measuring conformational dynamics in uniformly 13C-labeled proteins. The experiment was validated by using the E140Q mutant of the C-terminal fragment of calmodulin, which exhibits significant conformational exchange between two major conformations, as gauged from previous 15N and 1H relaxation studies. Consistent with previous work, the present 13C(alpha) R1rho experiment detects conformational-exchange dynamics throughout the protein. The average correlation time of <tau(ex)>=25+/-8 micros is in excellent agreement with those determined previously from 1H and 15N R1rho relaxation data: <tau(ex)>=19+/-7 and 21+/-3 micros, respectively. The extracted chemical-shift differences between the exchanging states reveal significant fluctuations in dihedral angles within single regions of Ramachandran phi-psi space, that were not identified from the 1H and 15N relaxation data. The present results underscore the advantage of using several types of nuclei to probe exchange dynamics in biomolecules.

Research paper thumbnail of Relaxation Dispersion NMR Spectroscopy as a Tool for Detailed Studies of Protein Folding

Biophysical Journal, 2009

Characterization of the mechanisms by which proteins fold into their native conformations is impo... more Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a ''highresolution'' picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

Research paper thumbnail of Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

Journal of Biomolecular NMR, 2009

A labeling scheme is introduced that facilitates the measurement of accurate (13)C(beta) chemical... more A labeling scheme is introduced that facilitates the measurement of accurate (13)C(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of (13)C enrichment (30-40%) at C(beta) side-chain carbon positions for 15 of the amino acids with little (13)C label at positions one bond removed (approximately 5%). A pair of samples are produced using [1-(13)C]-glucose/NaH(12)CO(3) or [2-(13)C]-glucose as carbon sources with isolated and enriched (>30%) (13)C(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of (13)C(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

Research paper thumbnail of Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

PLOS ONE, 2017

Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-te... more Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond timescale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.

Research paper thumbnail of A single-quantum methyl 13C-relaxation dispersion experiment with improved sensitivity

Journal of Biomolecular NMR, 2007

A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion pro... more A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.

Research paper thumbnail of Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

Journal of Biomolecular NMR, 2005

NMR spin relaxation in the rotating frame (R(1rho)) is a unique method for atomic-resolution char... more NMR spin relaxation in the rotating frame (R(1rho)) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R(1rho) relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R(1rho) data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 micros. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R(1rho) experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R(1rho) relaxation data, with average values of [see text] and 21 +/- 3 micros , respectively.

Research paper thumbnail of Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE

Proceedings of the National Academy of Sciences, 2010

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cyto... more MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae , with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for fu...

Research paper thumbnail of Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain

PLOS ONE, 2017

Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-... more Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-box and coiled-coil domains (RBCC), where the B-box domain is a unique feature of the TRIM protein family. TRIM21 is an E3 ubiquitin-protein ligase implicated in innate immune signaling by acting as an autoantigen and by modifying interferon regulatory factors. Here we report the three-dimensional solution structure of the TRIM21 B-box2 domain by nuclear magnetic resonance (NMR) spectroscopy. The structure of the B-box2 domain, comprising TRIM21 residues 86-130, consists of a short α-helical segment with an N-terminal short β-strand and two anti-parallel β-strands jointly found the core, and adopts a RINGlike fold. This ββαβ core largely defines the overall fold of the TRIM21 B-box2 and the coordination of one Zn 2+ ion stabilizes the tertiary structure of the protein. Using NMR titration experiments, we have identified an exposed interaction surface, a novel interaction patch where the B-box2 is likely to bind the N-terminal RING domain. Our structure together with comparisons with other TRIM B-box domains jointly reveal how its different surfaces are employed for various modular interactions, and provides extended understanding of how this domain relates to flanking domains in TRIM proteins.

Research paper thumbnail of Measurement of protein backbone 13CO and 15N relaxation dispersion at high resolution

Journal of Biomolecular NMR, 2017

Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics ... more Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates Maxim Mayzel and Alexandra Ahlner have contributed equally to this work.

Research paper thumbnail of Structural insights into the calcium-mediated allosteric transition in the C-terminal domain of calmodulin from NMR measurements

Biochemistry, Jan 30, 2015

Calmodulin is a two-domain signalling protein that becomes activated upon binding cooperatively t... more Calmodulin is a two-domain signalling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin function, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and NMR experiments on the Ca2+-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states, and validated such ensemble against three independent experimental datasets, namely inter-proton NOEs, 15N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the correspo...

Research paper thumbnail of Robust and convenient analysis of protein thermal and chemical stability

Protein Science, 2015

We present the software CDpal that is used to analyze thermal and chemical denaturation data to o... more We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.

Research paper thumbnail of Local destabilization of the metal-binding region in human copper-zinc superoxide dismutase by remote mutations is a possible determinant for progression of ALS

Biochemistry, Jan 20, 2015

More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismut... more More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to ...

Research paper thumbnail of Isotope Labeling Methods for Relaxation Measurements

Advances in Experimental Medicine and Biology, 2012

Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynami... more Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly (15)N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on (13)Cα, (13)Cβ or (13)CO or (1)Hα. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter.

Research paper thumbnail of Isotope Labeling Methods for Large Systems

Advances in Experimental Medicine and Biology, 2012

A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is th... more A major drawback of nuclear magnetic resonance (NMR) spectroscopy compared to other methods is that the technique has been limited to relatively small molecules. However, in the last two decades the size limit has been pushed upwards considerably and it is now possible to use NMR spectroscopy for structure calculations of proteins of molecular weights approaching 100 kDa and to probe dynamics for supramolecular complexes of molecular weights in excess of 500 kDa. Instrumental for this progress has been development in instrumentation and pulse sequence design but also improved isotopic labeling schemes that lead to increased sensitivity as well as improved spectral resolution and simplification. These are described and discussed in this chapter, focusing on labeling schemes for amide proton and methyl proton detected experiments. We also discuss labeling methods for other potentially useful positions in proteins.

Research paper thumbnail of Protein Conformational Dynamics by Relaxation Dispersion

Encyclopedia of Biophysics, 2013

Research paper thumbnail of Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches

PLoS computational biology, 2015

The process of resonance assignment is fundamental to most NMR studies of protein structure and d... more The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software...

Research paper thumbnail of Measuring the Signs of 1Hα Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R1ρ NMR Spectroscopy

Journal of the American Chemical Society, 2009

Research paper thumbnail of PINT: a software for integration of peak volumes and extraction of relaxation rates

Journal of Biomolecular NMR, 2013

We present the software PINT (Peak INTegration), designed to perform integration of peaks in NMR ... more We present the software PINT (Peak INTegration), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

Research paper thumbnail of Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

Journal of Biomolecular NMR, 2008

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectrosc... more Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated (13)C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (13)CO experiments however the large separation between (13)CO and (13)C(alpha) chemical shifts offers hope that robust (13)CO dispersion profiles can be recorded on uniformly (13)C labeled samples, leading to the extraction of accurate (13)CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-(13)C]-pyruvate and NaH(13)CO(3,) or uniformly labeled, generated from (13)C-glucose. Very similar (13)CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

Research paper thumbnail of Quantifying millisecond exchange dynamics in proteins by CPMG relaxation dispersion NMR using side-chain 1H probes

Journal of the American Chemical Society, Jan 15, 2012

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millise... more A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstra...

Research paper thumbnail of Microsecond Protein Dynamics Measured by 13Cα Rotating-Frame Spin Relaxation

ChemBioChem, 2005

NMR spin relaxation in the rotating frame (R1rho) is a unique method for atomic-resolution charac... more NMR spin relaxation in the rotating frame (R1rho) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond timescale. We present a rotating-frame 13C(alpha) relaxation dispersion experiment for measuring conformational dynamics in uniformly 13C-labeled proteins. The experiment was validated by using the E140Q mutant of the C-terminal fragment of calmodulin, which exhibits significant conformational exchange between two major conformations, as gauged from previous 15N and 1H relaxation studies. Consistent with previous work, the present 13C(alpha) R1rho experiment detects conformational-exchange dynamics throughout the protein. The average correlation time of <tau(ex)>=25+/-8 micros is in excellent agreement with those determined previously from 1H and 15N R1rho relaxation data: <tau(ex)>=19+/-7 and 21+/-3 micros, respectively. The extracted chemical-shift differences between the exchanging states reveal significant fluctuations in dihedral angles within single regions of Ramachandran phi-psi space, that were not identified from the 1H and 15N relaxation data. The present results underscore the advantage of using several types of nuclei to probe exchange dynamics in biomolecules.

Research paper thumbnail of Relaxation Dispersion NMR Spectroscopy as a Tool for Detailed Studies of Protein Folding

Biophysical Journal, 2009

Characterization of the mechanisms by which proteins fold into their native conformations is impo... more Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a ''highresolution'' picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

Research paper thumbnail of Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

Journal of Biomolecular NMR, 2009

A labeling scheme is introduced that facilitates the measurement of accurate (13)C(beta) chemical... more A labeling scheme is introduced that facilitates the measurement of accurate (13)C(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of (13)C enrichment (30-40%) at C(beta) side-chain carbon positions for 15 of the amino acids with little (13)C label at positions one bond removed (approximately 5%). A pair of samples are produced using [1-(13)C]-glucose/NaH(12)CO(3) or [2-(13)C]-glucose as carbon sources with isolated and enriched (>30%) (13)C(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of (13)C(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

Research paper thumbnail of Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

PLOS ONE, 2017

Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-te... more Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond timescale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.

Research paper thumbnail of A single-quantum methyl 13C-relaxation dispersion experiment with improved sensitivity

Journal of Biomolecular NMR, 2007

A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion pro... more A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.

Research paper thumbnail of Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

Journal of Biomolecular NMR, 2005

NMR spin relaxation in the rotating frame (R(1rho)) is a unique method for atomic-resolution char... more NMR spin relaxation in the rotating frame (R(1rho)) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R(1rho) relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R(1rho) data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 micros. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R(1rho) experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R(1rho) relaxation data, with average values of [see text] and 21 +/- 3 micros , respectively.