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Biomedical subjects

F M Poulsen

Publications and source records attributed to F M Poulsen.

At least 19 recordsLinked to original sources
PubMed2001

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.

Amino Acids↗
PubMed2000

Formation of hydrogen bonds precedes the rate-limiting formation of persistent structure in the folding of ACBP.

A burst phase in the early folding of the four-helix two-state folder protein acyl-coenzyme A binding protein (ACBP) has been detected using quenched-flow in combination with site-specific NMR-detected hydrogen exchange. Several of the burst phase structures coincide with a structure consisting of eight conserved hydrophobic residues at the interface between the two N and C-terminal helices. Previous mutation studies have shown that the formation of this structure is rate limiting for the final folding of ACBP. The burst phase structures observed in ACBP are different from the previously reported collapsed types of burst phase intermediates observed in the folding of other proteins.

Amides↗
PubMed1999

Acyl-coenzyme A binding protein (ACBP).

Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein and of the complex with palmitoyl-CoA using NMR spectroscopy. In the four alpha-helix bundle structure, a set of 21 highly conserved residues present in more that 90% of all known sequences of acyl-coenzyme A binding proteins constitutes three separate mini-cores. These residues are predominantly located at the helix-helix interfaces. From studies of a large set of mutant proteins the role of the conserved residues has been related to structure, function, folding and stability.

Amino Acid Sequence↗
PubMed1999

Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein.

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.

Acyl Coenzyme A↗
PubMed1999

Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone.

A set of three improved two-dimensional (2D) NMR methods for measuring one-bond (15)N-(1)H coupling constants in the protein backbone is presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D spectra into two separate subspectra corresponding to the two possible spin states of the coupling partner. Cross talk between the two subspectra is a second order effect in the difference between the actual coupling constants and the one used in setting the pertinent delays of the pulse sequences. This relatively high degree of editing accuracy makes the methods useful for applications to molecules subjected to weak alignment where the one-bond coupling constants are linear combinations of a scalar J and a residual dipolar contribution containing important structural information. A demonstration of the new methods is shown for the (15)N-labeled protein chymotrypsin inhibitor 2 in a lipid bicelle mixture.

Hydrogen↗
PubMed1999

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Amino Acid Sequence↗
PubMed1999

Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion.

The structure in solution of the second Ig-module fragment of residues 117-208 of NCAM has been determined. Like the first Ig-module of residues 20-116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been identified. The two-module fragment NCAM(20-208) is a stable dimer. Removal of the charged residues in these sites in NCAM(20-208) abolishes the dimerization. Modeling the dimer of NCAM(20-208) to fit the interactions of these charges produces one coherent binding site for the formation of two antiparallel strands of the first two NCAM modules. This mode of binding could be a major element in trans-cellular interactions in neural cell adhesion.

Amino Acid Substitution↗
PubMed1999

The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP.

The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24 probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding step and that a sequential framework model can describe the protein folding reaction.

Amino Acid Sequence↗
PubMed1998

Mapping the lifetimes of local opening events in a native state protein.

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.

Amides↗
PubMed1998

Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins.

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.

Antigens, Plant↗
PubMed1997

The solution structure of the N-terminal domain of alpha2-macroglobulin receptor-associated protein.

The three-dimensional structure of the N-terminal domain (residues 18-112) of alpha2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23-34, 39-65, and 73-88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix-loop-helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.

Animals↗
PubMed1997

Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands.

BACKGROUND: . Plant nonspecific lipid-transfer proteins (nsLTPs) bind a variety of very different lipids in vitro, including phospholipids, glycolipids, fatty acids and acyl coenzyme As. In this study we have determined the structure of a nsLTP complexed with palmitoyl coenzyme A (PCoA) in order to further our understanding of the structural mechanism of the broad specificity of these proteins and its relation to the function of nsLTPs in vivo. RESULTS: . 1H and 13C nuclear magnetic resonance spectroscopy (NMR) have been used to study the complex between a nsLTP isolated from barley seeds (bLTP) and the ligand PCoA. The resonances of 97% of the 1H atoms were assigned for the complexed bLTP and nearly all of the resonances were assigned in the bound PCoA ligand. The palmitoyl chain of the ligand was uniformly 13C-labelled allowing the two ends of the hydrocarbon chain to be assigned. The comparison of a subset of 20 calculated structures to an average structure showed root mean square deviations of 1.89 +/- 0.19 for all C, N, O, P and S atoms of the entire complex and of 0.57 +/- 0.09 for the peptide backbone atoms of the four alpha helices of the complexed bLTP. The four-helix topology of the uncomplexed bLTP is maintained in the complexed form of the protein. The bLTP only binds the hydrophobic parts of PCoA with the rest of the ligand remaining exposed to the solvent. The palmitoyl chain moiety of the ligand is placed in the interior of the protein and bent in a U-shape. This part of the ligand is completely buried within a hydrophobic pocket of the protein. CONCLUSIONS: . A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.

Amino Acid Sequence↗
PubMed1996

Time-resolved fluorescence studies of the molten globule state of apomyoglobin.

We have investigated the structure of the molten globule state of horse heart apomyoglobin by energy transfer experiments. The tyrosine residue at position 146 was converted into 3-nitro-tyrosine and distances between this side-chain and the two tryptophanyl side-chains were obtained from the time-resolved tryptophanyl fluorescence decay curve. Since both Trp residues are located in the N-terminal A-helix and the modified Tyr residue is located in the C-terminal H-helix, these measurements give information about this helix-helix distance. The energy transfer experiments provide direct evidence for a close contact between the A-helix and the H-helix in the molten globule state. This gives a very strong indication of the presence of a single near-native hydrophobic cluster in this state, as previously proposed by other authors. The distance distribution suggests that the fluctuations in the compact states have correlation times shorter than 1 ns. The experiments also show larger fluctuations, both in the native state and in the molten globule state. In addition, the tryptophanyl fluorescence anisotropy decay curves have been measured. The results suggest loose tertiary contacts in the molten globule state, which is in good agreement with earlier studies. For the denatured state of apomyoglobin, both techniques indicate an extended random coil structure.

Animals↗
PubMed1996

Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family.

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied. Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine. The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences. The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding. The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism.

Amino Acid Sequence↗
PubMed1996

Structure in solution of a four-helix lipid binding protein.

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.

Amino Acid Sequence↗