Biomedical subjects

Kaare Teilum

Publications and source records attributed to Kaare Teilum.

11 recordsLinked to original sources

PubMed2006

Solution structures of human and porcine beta-microseminoprotein.

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.

Amino Acid Sequence↗

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

Solution structure of human prolactin.

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

Amino Acid Sequence↗

PubMed2005

Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.

Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues.

Escherichia coli Proteins↗

PubMed2005

Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Biochemistry↗

PubMed2004

Determination of an ensemble of structures representing the denatured state of the bovine acyl-coenzyme a binding protein.

The denatured state of a protein contains important information about the determinants of the folding process. By combining site-directed spin-labeling NMR experiments and restrained computer simulations, we have determined ensembles of conformations that represent the denatured state of the bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride. As the experimentally determined distance information corresponds to weighted averages over a broad ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of conformations that are compatible with the experimental data and thus to obtain information regarding the distribution of structures in the denatured state. Our results show that the denatured state of ACBP is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests that the sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the structure in the denatured state of ACBP.

Acyl Coenzyme A↗

PubMed2002

Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding domain of flocculin, a cell-adhesion molecule from Saccharomyces carlsbergensis.

The recombinant carbohydrate-binding domain of the cell-surface lectin flocculin from brewer's yeast has been identified, purified and crystallized. The expression of the protein is associated with the nutritional state of the yeast. P2(1)2(1)2(1) crystals with unit-cell parameters a = 36.5, b = 59.7, c = 83.1 A were obtained in hanging drops at 295 K using 25%(w/v) PEG 4000, 0.05 M KH(2)PO(4) as precipitant. X-ray diffraction data have been obtained to 2.6 A. The asymmetric unit contains one molecule and has a solvent content of 32%. An isomorphous PtCl(4)(2-) derivative has been obtained.

Base Sequence↗

PubMed2002

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling.

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.

Circular Dichroism↗

PubMed2002

Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing.

Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue alpha-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 micros to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant tau = 80 micros, indicating formation of an intermediate with only slightly enhanced fluorescence of Trp-55 and Trp-58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C terminus by labeling an I86C mutant of ACBP with 5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid]. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-micros time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with approximately 1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms.

Amino Acid Substitution↗

PubMed2002

Transient intermediary states with high and low folding probabilities in the apparent two-state folding equilibrium of ACBP at low pH.

Measurements of the stability as a function of pH for the acyl-coenzyme A binding protein (ACBP) has shown a significant difference in the pH transition midpoint measured by NMR spectroscopy at pH 3.12 and the transition midpoint measured at pH 2.92 and 2.97 by circular dichroism and by fluorescence spectroscopy, respectively. A similar behavior has not been observed in other proteins. It is suggested that these differences arise because the population of the unfolded molecules still contains significant amounts of native like secondary and tertiary structure. NMR spectroscopy measures the concentration of the two components of the folding unfolding equilibrium individually, whereas circular dichroism and fluorescence measure the concentration of the conformations of the light-absorbing chromophores present in both the folded and the unfolded molecules. In the narrow pH range, nascent structure can be detected as the average amount of secondary structure per unfolded molecule and hydrophobic interactions in the population of unfolded molecules. These structures are not observable immediately by NMR spectroscopy; however, a chemical shift analysis of the peptide backbone (13)C chemical shift indicates strongly the existence of short-lived and transient helical structures at pH 2.3. Magnetization transfer studies have been applied to study the equilibrium between folded and unfolded ACBP near the pH transition point measured by NMR. This study has shown that there are two categories of subpopulations in the population of unfolded ACBP. One for which magnetization can be transferred to the folded form during the folding process, and one for which transfer is not observed. The molecules of the latter population of unfolded protein apparently, do not fold within the time-frame of the magnetization transfer experiment. This result suggests the existence of a subpopulation of the acid-unfolded protein molecules with a high propensity for folding. It is suggested that in this subpopulation, a particular set of native like interactions in the peptide backbone and between side-chains in the peptide chain have to be formed.

Animals↗