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L Sjölin

Publications and source records attributed to L Sjölin.

At least 19 recordsLinked to original sources
PubMed2001

Structure of Escherichia coli fragment TR2C from calmodulin to 1.7 A resolution.

Fragment TR2C is the C-terminal part of the calcium-binding protein calmodulin, including residues 78-148. The crystal structure of TR2C was solved by molecular replacement and refined to a conventional R value of 21.8% (R(free) = 22.0%), using all data in the resolution range 20.0-1.7 A. This study shows that the secondary structure of TR2C, a pair of EF-hand motifs with two calcium-binding sites, is similar to the corresponding motifs in intact calmodulin. However, it also indicates that the N-terminus of helix E is closer to the C-terminus of helix H in TR2C than in the intact protein and that the loop connecting the EF-hands shows different conformations in the two structures. The crystal structure of TR2C was further found to be similar to the set of NMR structures of this fragment, although some pronounced differences exist.

Calmodulin↗
PubMed2000

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.

Azurin↗
PubMed1998

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex. RESULTS: We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites. CONCLUSIONS: This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Binding Sites↗
PubMed1997

X-ray structure of a ribonuclease A-uridine vanadate complex at 1.3 A resolution.

The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 A resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjölin (1983). Proc. Natl Acad. Sci. USA, 80, 3628-3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V-O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjölin & Gilliland (1988). Biochemistry, 27, 2705-2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.

Journal Article↗
PubMed1997

X-ray structure determination and characterization of the Pseudomonas aeruginosa azurin mutant Met121Glu.

The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.

Azurin↗
PubMed1997

The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the Co(II) derivative and Co-EPR spectroscopy.

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial.

Azurin↗
PubMed1996

Mutant Met121Ala of Pseudomonas aeruginosa azurin and its azide derivative: crystal structures and spectral properties.

The crystal structures of the azurin mutant Met121Ala and its azide derivative Met121Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met121Ala mutant, the distance between the copper ion and His117 increases by 0.34 A compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74 A. In the Met121Ala-azide structure, the azide anion occupies the cavity created by replacing the Met121 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met121Ala mutant still maintains the properties of an ordinary type 1 copper site while the Met121Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

Journal Article↗
PubMed1995

Structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa at 2.2 A resolution.

The structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa has been determined by difference Fourier synthesis using phases from the wild-type azurin model. The final crystallographic R value is 0.170 for 17 394 reflections to a resolution of 2.2 A. The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1), a = 57.4, b = 80.4, c = 110.3 A. The four molecules in the asymmetric unit are packed as a dimer of dimers. The nickel metal site of this mutant structure is similar to the zinc metal site in the azurin Asp47 mutant. The site-specific mutation was performed at residue Trp48, which is located in the center of the protein in a highly hydrophobic environment, to investigate its suggested role in the long-range electron-transfer pathway between the disulfide bond on one side of the protein to the Cu centre. The structure around the mutation site Met48 showed no significant change compared with the wild-type structure.

Journal Article↗
PubMed1995

Structure of the azurin mutant Phe114Ala from Pseudomonas aeruginosa at 2.6 A resolution.

The crystal structure of azurin mutant Phe114Ala from Pseudomonas aeruginosa has been solved by molecular replacement. The final crystallographic R value is 0.185 for 9832 reflections to a resolution of 2.6 A. The root-mean-square deviation for main-chain atom positions is 0.020 A between the four independent monomers in the asymmetric unit. The mutant Ala114 crystallized from PEG 4000 in a new crystal form and the crystals are monoclinic, P2(1), a= 51.0, b = 83.6, c= 66.4 A and beta = 110.5 degrees. The four molecules in the asymmetric unit are packed as a dimer of dimers and are related by an approximate twofold axis. The dimer packing and the dimer contact region are very similar to that of the Alcaligenes denitrificans azurin dimer. The mutation was performed at residue Phe114, which exhibits a pi-electron overlap with the copper ligand His117, to investigate its suggested role in the electron self-exchange reaction. Removal of steric constrains from the phenylalanine side chain created a somewhat different geometry around the copper site with an increased mobility of His117 resulting in an enlarged Cu-N length which may be responsible for the slight differences obtained in the spectral properties of the mutant versus the wild-type protein.

Journal Article↗
PubMed1993

Structure of Pseudomonas aeruginosai zinc azurin mutant Asn47Asp at 2.4 A resolution.

The Pseudomonas aeruginosa azurin mutant Asn47Asp has been isolated, its spectroscopic and kinetic properties characterized, and the X-ray crystal structure of its zinc derivative determined. While the optical and electron paramagnetic resonance spectra as well as the electron-transfer activity of the mutant are very similar to the wild-type values, the Asn47Asp reduction potential is slightly increased by 20 mV. The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 57.8, b = 81.5 and c = 112.6 A. There are four molecules in the asymmetric unit, packed as a tetramer which consists of two independent dimers. The zinc site of this mutant structure is similar to the wild-type zinc azurin and, in particular, the metal-binding site is almost identical to the site found in the wild-type zinc-azurin structure [Nar, Huber, Messerschmidt, Filippou, Barth, Jaquinod, Kamp & Canters (1992). Eur. J. Biochem. 205, 1123-1129]. The Asp47 side chain at that mutation site takes on a very similar orientation to Asn47 in the wild-type structure preserving the two hydrogen bonds with the neighbouring Thr113 NH and O(gamma)H. Therefore, the increased reduction potential of the mutant is probably a result of an altered charge distribution close to the metal site.

Journal Article↗
PubMed1993

Experience with phase extension and ab initio phase determination in macromolecular crystallography using maximum-entropy methods.

Three procedures, or 'tools', have been developed and tested for applying maximum-entropy methods to phase extension and to ab initio phase determination. The phase expander tool has been used in connection with the solution of two previously unknown macromolecular structures. An efficient algorithm for the determination of an electron-density distribution that is everywhere positive and that agrees with observed structure amplitudes (tools II and III) has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain, the structure of which was recently determined using replacement methods. By use of the same maximum-entropy methods, the structure amplitudes from the unknown structure of bovine heart creatine kinase, a protein with 381 amino-acid residues, have been phased ab initio to 2.7 A resolution. The phases of the centric reflections have also been confirmed by a satisfactory solution of the Patterson map of a mercury derivative. The current status of the structure interpretation is presented. This technique has also been applied to a test case where 48 centric reflections from bovine prothrombin fragment 1 data were phased ab initio and subsequently used in the determination of Patterson solutions for a heavy-atom derivative data set.

Journal Article↗
PubMed1993

3D-structural elucidation of biologically important macromolecules.

In 1953 Max Perutz discovered that the phase problem of protein crystallography could be solved by the method of isomorphous replacement with heavy atoms. At that time scientists expected that the structure, not only of haemoglobin, but also of many other proteins, would soon be solved. This did not happen. Only three protein structures had been solved by 1965, and only eleven by 1970. The practical difficulties of crystallization and data collection and, in addition, the enormous effort to interpret an electron density map at that time, made scientists reluctant to enter this risky new field. Today, the situation is transformed and more than 400 protein structures have been solved. Many of these structures are of practical interest to medicine, an interest that often becomes apparent after the solution of the protein. This paper is a brief review of the techniques being used in the field of crystallographic elucidation of macromolecular structures. In particular, some aspects of the structures of ribonuclease A, calmodulin and the human growth hormone-receptor complex are presented. These structures serve as examples and thus they represent the significance of the results which are normally obtained from a regular crystallographic investigation.

Macromolecular Substances↗
PubMed1992

Crystallization and preliminary crystallographic data for the azurin mutant Ala 114 from Pseudomonas aeruginosa.

The site-specific mutant alanine 114 of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli has been crystallized from PEG 4000 in a new crystal form compared to the wild type utilizing the hanging-drop procedure. The crystals are blue well-formed prisms. Monoclinic, P2(1), a = 51.03 (5), b = 83.36 (5), c = 66.30 (6) A and beta = 111.0 (1) degrees. 14,875 reflections up to 2.7 A have been collected using a modified Syntex P2(1) automated four-circle diffractometer.

Azurin↗
PubMed1991

Ab initio phase determination for X-ray diffraction data from crystals of a native protein.

An efficient algorithm for the determination of an everywhere positive electron-density distribution that agrees with observed structure amplitudes has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain whose structure was recently determined using molecular replacement methods. A systematic procedure for testing the signs of centric reflections, using the total entropy of the map as a figure of merit, was used to produce a low-resolution map. The phases of acentric and additional centric reflections were then chosen by adding them to the map with various possible phases and computing the total entropy of the resulting map. Of 159 centric reflections whose phases were chosen by this procedure, 141 had the same phase as in the refined structure. The median absolute phase difference for 1811 acentric reflections was 32 degrees. A map produced from these 1970 reflections, out of 12,346 reflections in the data set, showed a remarkable agreement with the refined structure. This molecule is many times larger than any whose structures have previously been determined without the use of isomorphous replacement, molecular replacement or anomalous dispersion, and the map demonstrates the potential of maximum-entropy methods in macromolecular structure determination.

Algorithms↗
PubMed1991

Cassette mutagenesis of Met121 in azurin from Pseudomonas aeruginosa.

Cassette mutagenesis was used to exchange the suggested copper ligand Met121 in azurin to all other amino acids, and a stop codon. The mutant proteins were characterized by optical absorption spectroscopy and EPR. At low pH, all mutants exhibit the characteristics of a blue type 1 copper protein, indicating that methionine is not needed to create a blue copper site. At high pH, the Glu121 and the Lys121 mutants constitute a new form of protein-bound copper that is outside the range of type 1 copper.

Azurin↗
PubMed1988

Analysis of solvent structure and hydrogen exchange in proteins on the basis of neutron diffraction data from deuterated and hydrogenous crystals.

A method has been developed to determine the structure of bound solvent and the positions of exchanged hydrogens in proteins, on the basis of neutron diffraction from hydrogenous and deuterated crystals. In this method phases for the hydrogenous and for the deuterated model are refined simultaneously, and an average model is imposed in the volume occupied by non-hydrogen atoms. The densities in the areas of bulk solvent are replaced by their average values, while no modifications are performed in the vicinity of ordered solvents and potentially exchangeable hydrogens. The method was tested on 1.8 A neutron diffraction data collected from two crystals of bovine pancreatic trypsin inhibitor, one of them deuterated and the other hydrogenous. Significant improvement was observed for the densities corresponding to many partially occupied solvent sites, as well as to partially exchanged hydrogens. The algorithm presented here has been compared with a different approach published recently by Shpungin & Kossiakoff [Methods Enzymol.(1986), 127, 329-342].

Amides↗
PubMed1988

Structure of phosphate-free ribonuclease A refined at 1.26 A.

The structure of phosphate-free bovine ribonuclease A has been refined at 1.26-A resolution by a restrained least-squares procedure to a final R factor of 0.15. X-ray diffraction data were collected with an electronic position-sensitive detector. The final model consists of all atoms in the polypeptide chain including hydrogens, 188 water sites with full or partial occupancy, and a single molecule of 2-methyl-2-propanol. Thirteen side chains were modeled with two alternate conformations. Major changes to the active site include the addition of two waters in the phosphate-binding pocket, disordering of Gln-11, and tilting of the imidazole ring of His-119. The structure of the protein and of the associated solvent was extensively compared with three other high-resolution, refined structures of this enzyme.

Binding Sites↗