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Mikael Akke

Publications and source records attributed to Mikael Akke.

At least 19 recordsLinked to original sources
PubMed2006

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain.

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.

Conserved Sequence↗
PubMed2006

Functional dynamics of human FKBP12 revealed by methyl 13C rotating frame relaxation dispersion NMR spectroscopy.

Transverse relaxation dispersion NMR spectroscopy can provide atom-specific information about time scales, populations, and the extent of structural reorganization in proteins under equilibrium conditions. A method is described that uses side-chain methyl groups as local reporters for conformational transitions taking place in the microsecond regime. The experiment measures carbon nuclear spin relaxation rates in the presence of continuous wave off-resonance irradiation, in proteins uniformly enriched with 13C, and partially randomly labeled with 2H. The method was applied to human FK-506 binding protein (FKBP12), which uses a common surface for binding substrates in its dual role as both an immunophilin and folding assistant. Conformational dynamics on a time scale of approximately 130 micros were detected for methyl groups located in the substrate binding pocket, demonstrating its plasticity in the absence of substrate. The spatial arrangement of affected side-chain atoms suggests that substrate recognition involves the rapid relative movement of the subdomain comprising residues Ala81-Thr96 and that the observed dynamics play an important role in facilitating the interaction of this protein with its many partners, including calcineurin.

Carbon Isotopes↗
PubMed2006

The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein.

The folding kinetics of bovine acyl-CoA binding protein was studied by 15N relaxation dispersion measurements under equilibrium conditions. Relaxation dispersion profiles were measured at several concentrations of guanidine hydrochloride (GuHCl). The unfolding rate constant (k(u)) was determined under conditions favoring folding, for which the folding rate constant (k(f)) dominates the relaxation in stopped-flow kinetic measurements. Conversely, k(f) was determined under conditions favoring unfolding, for which k(u) dominates stopped-flow data. The rates determined by NMR therefore complement those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of k(f) and k(u) in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models for folding. However, although the values of k(f) determined by the two methods agree, the values of k(u) do not. As a result, a combined analysis of all data does not comply with a two-state model but indicates that an unfolding intermediate exists on the native side of the dominant energy barrier. The denaturant and temperature dependencies of the chemical shifts and k(u) indicate that the intermediate state is structurally similar to the native state. Equilibrium unfolding monitored by optical spectroscopy corroborate these conclusions. The temperature dependence of the chemical shifts identifies regions of the protein that are selectively destabilized in the intermediate. These results illustrate the power of combining stopped-flow kinetics and NMR spectroscopy to analyze protein folding.

Anilino Naphthalenesulfonates↗
PubMed2006

Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements.

Site-specific 13C labeling offers a desirable means of eliminating unwanted relaxation pathways and coherent magnetization transfer in NMR relaxation experiments. Here we use [1-13C]-glucose as the sole carbon source in the growth media for protein overexpression in Escherichia coli. The approach results in specific incorporation of 13C at isolated positions in the side chains of aromatic amino acids, which greatly simplifies the measurements and interpretation of 13C relaxation rates in these spin systems. The method is well suited for characterization of chemical exchange by CPMG or spin-lock relaxation methods. We validated the method by acquiring 13C rotating-frame relaxation dispersion data on the E140Q mutant of the C-terminal domain of calmodulin, which reveal conformational exchange dynamics with a time constant of 71 mus for Y138.

Acyl Coenzyme A↗
PubMed2005

Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins.

Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used (13)C(alpha)(i - 1)/(13)C(alpha)(i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc.), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of approximately 20 micros. The present results reveal that an entire alpha-helix undergoes partial unraveling in a transient and cooperative manner.

Amino Acids↗
PubMed2005

Compact oleic acid in HAMLET.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex between alpha-lactalbumin and oleic acid that induces apoptosis in tumor cells, but not in healthy cells. Heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of 13C-oleic acid in HAMLET, and to study the 15N-labeled protein. Nuclear Overhauser enhancement spectroscopy shows that the two ends of the fatty acid are in close proximity and close to the double bond, indicating that the oleic acid is bound to HAMLET in a compact conformation. The data further show that HAMLET is a partly unfolded/molten globule-like complex under physiological conditions.

Humans↗
PubMed2005

Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride.

We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (muHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol(-1) and -2.6 kcal mol(-1) for GdnHCl and AOT, respectively. Hence, the AOT denatured state (D(AOT)) is less destabilised than the GdnHCl denatured state (D(GdnHCl)), relative to the native state in water. Far-UV CD spectroscopy revealed that D(AOT) retains some secondary structure, while D(GdnHCl) is essentially unstructured. Similarly, fluorescence data suggest that D(AOT) is more compact than D(GdnHCl). Activity measurements reveal that both D(AOT) and D(GdnHCl) are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of D(AOT) in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of D(AOT) in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data.

Amino Acid Sequence↗
PubMed2005

Microsecond protein dynamics measured by 13Calpha rotating-frame spin relaxation.

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 =25+/-8 micros is in excellent agreement with those determined previously from 1H and 15N R1rho relaxation data: =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.

Carbon Isotopes↗
PubMed2005

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins.

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.

Ammonium Chloride↗
PubMed2004

Transient aggregation and stable dimerization induced by introducing an Alzheimer sequence into a water-soluble protein.

Transient contacts between denatured polypeptide chains are likely to play an important part in the initial stages of protein aggregation and fibrillation. To analyze the nature of such contacts, we have carried out a protein engineering study of the 102-residue protein U1A, which aggregates transiently in the wild-type form during refolding from the guanidinium chloride-denatured state. We have prepared a series of mutants with increased aggregation tendencies by increasing the homology between two beta-strands of U1A and the Alzheimer peptide (beta-AP). These mutants undergo transient aggregation during refolding, as measured by concentration dependence, double-jump experiments, and binding of ANS, a probe for exposed hydrophobic patches on protein surfaces. The propensity to aggregate increases with increasing homology to beta-AP. Further, the degree of transient ANS binding correlates reasonably well with the structural parameters recently shown to play a role in the fibrillation of natively unfolded proteins. Two mutants highly prone to transient aggregation, U1A-J and U1A-G, were also studied by NMR. Secondary structural elements of the U1A-J construct (with lower beta-AP homology) are very similar to those observed in U1A-wt. In contrast, the high-homology construct U1A-G exhibits local unfolding of the C-terminal helix, which packs against the beta-sheet in the wild-type protein. U1A-G is mainly dimeric according to (15)N spin relaxation data, and the dimer interface most likely involves the beta-sheet. Our data suggest that the transient aggregate relies on specific intermolecular interactions mediated by structurally flexible regions and that contacts may be formed in different beta-strand registers.

Alzheimer Disease↗
PubMed2004

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome.

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.

Dimerization↗
PubMed2004

Quantitative analysis of conformational exchange contributions to 1H-15N multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant.

Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential 1H-15N multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 +/- 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the 1H and 15N chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential 1H-15N multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from 15N off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the 1H and 15N spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the 1H chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on 1H single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.

Calmodulin↗
PubMed2004

Out of hot water.

Explore the source record for details and available documents.

Adenylate Kinase↗
PubMed2003

Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution.

We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse (15)N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide (15)N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.

Animals↗
PubMed2003

A statistical analysis of NMR spectrometer noise.

Estimation of NMR spectral parameters, using e.g. maximum likelihood methods, is commonly based on the assumption of white complex Gaussian noise in the signal obtained by quadrature detection. Here we present a statistical analysis with the purpose of discussing and testing the validity of this fundamental assumption. Theoretical expressions are derived for the correlation structure of the noise under various conditions, showing that in general the noise in the sampled signal is not strictly white, even if the thermal noise in the receiver steps prior to digitisation can be characterised as white Gaussian noise. It is shown that the noise correlation properties depend on the ratio between the sampling frequency and the filter cut-off frequency, as well as the filter characteristics. The theoretical analysis identifies conditions that are expected to yield non-white noise in the sampled signal. Extensive statistical characterisation of experimental noise confirms the theoretical predictions. The statistical methods outlined here are also useful for residual analysis in connection with validation of the model and the parameter estimates.

Magnetic Resonance Spectroscopy↗
PubMed2002

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.

Animals↗
PubMed2002

Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.

Calbindin D(28k) is a member of the calmodulin superfamily of Ca(2+)-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca(2+) buffer, but the studies presented in this work indicate that it may also act as a Ca(2+) sensor. The results show that Mg(2+) binds to the same sites as Ca(2+) with an association constant of approximately 1.4.10(3) m(-1) in 0.15 m KCl. The four high affinity sites in calbindin D(28k) bind Ca(2+) in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg(2+), the Ca(2+) affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca(2+) concentration in a resting cell calbindin D(28k) is saturated to 40-75% with Mg(2+) but to less than 9% with Ca(2+). In contrast, the protein is expected to be nearly fully saturated with Ca(2+) at the Ca(2+) level of an activated cell. A substantial conformational change is observed upon Ca(2+) binding, but only minor structural changes take place upon Mg(2+) binding. This suggests that calbindin D(28k) undergoes Ca(2+)-induced structural changes upon Ca(2+) activation of a cell. Thus, calbindin D(28k) displays several properties that would be expected for a protein involved in Ca(2+)-induced signal transmission and hence may function not only as a Ca(2+) buffer but also as a Ca(2+) sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca(2+), becomes exposed upon Ca(2+) binding.

Amino Acid Sequence↗