Biomedical subjects

A O Smalås

Publications and source records attributed to A O Smalås.

At least 19 recordsLinked to original sources

PubMed2001

Crystallization and preliminary X-ray diffraction analysis of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua).

Uracil-DNA glycosylase (UDG) is a DNA-repair enzyme involved in the removal of uracil from DNA. The Atlantic cod UDG (cUDG) possesses typical cold-adaptation features, with higher catalytic efficiency and lower thermal stability than the mammalian counterparts. cUDG has been crystallized by the vapour-diffusion method using sodium citrate as the precipitant at pH 7.5. The crystals are monoclinic and belong to space group P2(1), with unit-cell parameters a = 68.58, b = 67.19, c = 68.64 A, beta = 119.85 degrees. There are two molecules in the asymmetric unit, with a corresponding V(M) value of 2.71 A(3) Da(-1) and a solvent content of 54.7%. Synchrotron diffraction data have been collected to 1.9 A resolution using cryogenic conditions (120 K).

Animals↗

PubMed2001

Electrostatic effects play a central role in cold adaptation of trypsin.

Organisms that live in constantly cold environments have to adapt their metabolism to low temperatures, but mechanisms of enzymatic adaptation to cold environments are not fully understood. Cold active trypsin catalyses reactions more efficiently and binds ligands more strongly in comparison to warm active trypsin. We have addressed this issue by means of comparative free energy calculations studying the binding of positively charged ligands to two trypsin homologues. Stronger inhibition of the cold active trypsin by benzamidine and positively charged P1-variants of BPTI is caused by rather subtle electrostatic effects. The different affinity of benzamidine originates solely from long range interactions, while the increased binding of P1-Lys and -Arg variants of BPTI is attributed to both long and short range effects that are enhanced in the cold active trypsin compared to the warm active counterpart. Electrostatic interactions thus provide an efficient strategy for cold adaptation of trypsin.

Adaptation, Physiological↗

PubMed2001

Atomic resolution structures of trypsin provide insight into structural radiation damage.

Radiation damage is an inherent problem in protein X-ray crystallography and the process has recently been shown to be highly specific, exhibiting features such as cleavage of disulfide bonds, decarboxylation of acidic residues, increase in atomic B factors and increase in unit-cell volume. Reported here are two trypsin structures at atomic resolution (1.00 and 0.95 A), the data for which were collected at a third-generation synchrotron (ESRF) at two different beamlines. Both trypsin structures exhibit broken disulfide bonds; in particular, the bond from Cys191 to Cys220 is very sensitive to synchrotron radiation. The data set collected at the most intense beamline (ID14-EH4) shows increased structural radiation damage in terms of lower occupancies for cysteine residues, more breakage in the six disulfide bonds and more alternate conformations. It appears that high intensity and not only the total X-ray dose is most harmful to protein crystals.

Animals↗

PubMed2001

Computational analysis of binding of P1 variants to trypsin.

The binding of P1 variants of bovine pancreatic trypsin inhibitor (BPTI) to trypsin has been investigated by means of molecular dynamics simulations. The specific interaction formed between the amino acid at the primary binding (P1) position of the binding loop of BPTI and the specificity pocket of trypsin was estimated by use of the linear interaction energy (LIE) method. Calculations for 13 of the naturally occurring amino acids at the P1 position were carried out, and the results obtained were found to correlate well with the experimental binding free energies. The LIE calculations rank the majority of the 13 variants correctly according to the experimental association energies and the mean error between calculated and experimental binding free energies is only 0.38 kcal/mole, excluding the Glu and Asp variants, which are associated with some uncertainties regarding protonation and the possible presence of counter-ions. The three-dimensional structures of the complex with three of the P1 variants (Asn, Tyr, and Ser) included in this study have not at present been solved by any experimental techniques and, therefore, were modeled on the basis of experimental data from P1 variants of similar size. Average structures were calculated from the MD simulations, from which specific interactions explaining the broad variation in association energies were identified. The present study also shows that explicit treatment of the complex water-mediated hydrogen bonding network at the protein-protein interface is of crucial importance for obtaining reliable binding free energies. The successful reproduction of relative binding energies shows that this type of methodology can be very useful as an aid in rational design and redesign of biologically active macromolecules.

Animals↗

PubMed2000

Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases.

The role of the S(1) subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P(1) position of the reactive site. The effects of substitutions at the P(1) position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala>Gly>Ser>Arg>Val>Leu>Trp. Calorimetric and circular dichroism methods showed that none of the P1 substitutions, except the P1-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine beta-trypsin and P(1)-Leu BPTI showed that the P(1)-Leu sterically conflicts with the side-chain of P(3)-Ile, which thereby is forced to rotate approximately 90 degrees. Ile18 (P(3)) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1' position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15 degrees compared to the native trypsin-BPTI complex.

Amino Acid Substitution↗

PubMed2000

Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: qualitative evaluation of electrostatic differences at the substrate binding site.

A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson-Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S(1) region of all trypsins is generated from Asp(189), but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S(1)-pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S(1)-pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S(1) pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes.

Amino Acid Sequence↗

PubMed2000

Cold adapted enzymes.

The number of reports on enzymes from cold adapted organisms has increased significantly over the past years, and reveals that adaptive strategies for functioning at low temperature varies among enzymes. However, the high catalytic efficiency at low temperature seems, for the majority of cold active enzymes, to be accompanied by a reduced thermal stability. Increased molecular flexibility to compensate for the low working temperature, is therefore still the most dominating theory for cold adaptation, although there also seem to be other adaptive strategies. The number of experimentally determined 3D structures of enzymes possessing cold adaptation features is still limited, and restricts a structural rationalization for cold activity. The present summary of structural characteristics, based on comparative studies on crystal structures (7), homology models (7), and amino acid sequences (24), reveals that there are no common structural feature that can account for the low stability, increased catalytic efficiency, and proposed molecular flexibility. Analysis of structural features that are thought to be important for stability (e.g. intra-molecular hydrogen bonds and ion-pairs, proline-, methionine-, glycine-, or arginine content, surface hydrophilicity, helix stability, core packing), indicates that each cold adapted enzyme or enzyme system use different small selections of structural adjustments for gaining increased molecular flexibility that in turn give rise to increased catalytic efficiency and reduced stability. Nevertheless, there seem to be a clear correlation between cold adaptation and reduced number of interactions between structural domains or subunits. Cold active enzymes also seem, to a large extent, to increase their catalytic activity by optimizing the electrostatics at and around the active site.

Adaptation, Biological↗

PubMed2000

Structural comparison of psychrophilic and mesophilic trypsins. Elucidating the molecular basis of cold-adaptation.

Structural rationalizations for differences in catalytic efficiency and stability between mesophilic and cold-adapted trypsins have been suggested from a detailed comparison of eight trypsin structures. Two trypsins, from Antarctic fish and Atlantic cod, have been constructed by homology modeling techniques and compared with six existing X-ray structures of both cold-adapted and mesophilic trypsins. The structural analysis focuses on the cold trypsin residue determinants found in a more extensive comparison of 27 trypsin sequences, and reveals a number of structural features unique to the cold-adapted trypsins. The increased substrate affinity of the psychrophilic trypsins is probably achieved by a lower electrostatic potential of the S1 binding pocket particularly arising from Glu221B, and from the lack of five hydrogen bonds adjacent to the catalytic triad. The reduced stability of the cold trypsins is expected to arise from reduced packing in two distinct core regions, fewer interdomain hydrogen bonds and from a destabilized C-terminal alpha-helix. The helices of the cold trypsins lack four hydrogen bonds and two salt-bridges, and they have poorer van der Waals packing interactions to the body of the molecule, compared to the mesophilic counterparts.

Adaptation, Physiological↗

PubMed2000

Evaluation of protein-protein association energies by free energy perturbation calculations.

The association energy upon binding of different amino acids in the specificity pocket of trypsin was evaluated by free energy perturbation calculations on complexes between bovine trypsin (BT) and bovine pancreatic trypsin inhibitor (BPTI). Three simulations of mutations of the primary binding residue (P(1)) were performed (P(1)-Ala to Gly, P(1)-Met to Gly and P(1)-Met to Ala) and the resulting differences in association energy (DeltaDeltaG(a)) are 2. 28, 5.08 and 2.93 kcal/mol for P(1)-Ala to Gly, P(1)-Met to Gly and to Ala with experimental values of 1.71, 4.62 and 2.91 kcal/mol, respectively. The calculated binding free energy differences are hence in excellent agreement with the experimental binding free energies. The binding free energies, however, were shown to be highly dependent on water molecules at the protein-protein interface and could only be quantitatively estimated if the correct number of such water molecules was included. Furthermore, the cavities that were formed when a large amino acid side-chain is perturbed to a smaller one seem to create instabilities in the systems and had to be refilled with water molecules in order to obtain reliable results. In addition, if the protein atoms that were perturbed away were not replaced by water molecules, the simulations dramatically overestimated the initial state of the free energy perturbations.

Alanine↗

PubMed1999

The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI.

The high-resolution X-ray structures have been determined for ten complexes formed between bovine beta-trypsin and P1 variants (Gly, Asp, Glu, Gln, Thr, Met, Lys, His, Phe, Trp) of bovine pancreatic trypsin inhibitor (BPTI). All the complexes were crystallised from the same conditions. The structures of the P1 variants Asp, Glu, Gln and Thr, are reported here for the first time in complex with any serine proteinase. The resolution of the structures ranged from 1.75 to 2.05 A and the R-factors were about 19-20 %. The association constants of the mutants ranged from 1.5x10(4) to 1.7x10(13) M-1. All the structures could be fitted into well-defined electron density, and all had very similar global conformations. All the P1 mutant side-chains could be accomodated at the primary binding site, but relative to the P1 Lys, there were small local changes within the P1-S1 interaction site. These comprised: (1) changes in the number and dynamics of water molecules inside the pocket; (2) multiple conformations and non-optimal dihedral angles for some of the P1 side-chains, Ser190 and Gln192; and (3) changes in temperature factors of the pocket walls as well as the introduced P1 side-chain. Binding of the cognate P1 Lys is characterised by almost optimal dihedral angles, hydrogen bonding distances and angles, in addition to considerably lower temperature factors. Thus, the trypsin S1 pocket seems to be designed particularly for lysine binding.

Amino Acid Substitution↗

PubMed1999

High-resolution structures of three new trypsin-squash-inhibitor complexes: a detailed comparison with other trypsins and their complexes.

An anionic trypsin from Atlantic salmon and bovine trypsin have been complexed with the squash-seed inhibitors, CMTI-I (Cucurbita maxima trypsin inhibitor I, P1 Arg) and CPTI-II (Cucurbita pepo trypsin inhibitor II, P1 Lys). The crystal structures of three such complexes have been determined to 1.5-1.8 A resolution and refined to crystallographic R factors ranging from 17.6 to 19.3%. The two anionic salmon-trypsin complexes (ST-CPTI and ST-CMTI) and the bovine-trypsin complex (BT-CPTI) have been compared to other trypsin-inhibitor complexes by means of general structure and primary and secondary binding features. In all three new structures, the primary binding residue of the inhibitor binds to trypsin in the classical manner, but with small differences in the primary and secondary binding patterns. Lysine in CPTI-II binds deeper in the specificity pocket of bovine trypsin than lysine in other known lysine-bovine-trypsin complexes, and anionic salmon trypsin lacks some of the secondary binding interactions found in the complexes formed between squash inhibitors and bovine trypsin. The ST-CMTI complex was formed from the reactive-site-cleaved form of the inhibitor. However, well defined electron density was observed for the P1-P1' peptide bond, together with a hydrogen-bonding pattern virtually identical to those of all serine-protease-protein-inhibitor complexes, indicating a resynthesis of the scissile bond.

Amino Acid Sequence↗

PubMed1999

Residue determinants and sequence analysis of cold-adapted trypsins.

The digestive enzyme trypsin is among the most extensively studied proteins, and its structure has been reported from a large number of organisms. This article focuses on the trypsins from vertebrates adapted to life at low temperatures. Cold-adapted organisms seem to have compensated for the reduced reaction rates at low temperatures by evolving more active and less temperature-stable enzymes. We have analyzed 27 trypsin sequences from a variety of organisms to find unique attributes for the cold-adapted trypsins, comparing trypsins from salmon, Antarctic fish, cod, and pufferfish to other vertebrate trypsins. Both the "cold" and the "warm" active trypsins have about 50 amino acids that are unique and conserved within each class. The main unique features of the cold-adapted trypsins attributable to low-temperature adaptation seem to be (1) reduced hydrophobicity and packing density of the core, mainly because of a lower (Ile + Leu)/(Ile + Leu + Val) ratio, (2) reduced stability of the C-terminal, (3) lack of one warm trypsin conserved proline residue and one proline tyrosine stacking, (4) difference in charge and flexibility of loops extending the binding pocket, and (5) different conformation of the "autolysis" loop that is likely to be involved in substrate binding.

Amino Acid Sequence↗

PubMed1999

Comparative molecular dynamics of mesophilic and psychrophilic protein homologues studied by 1.2 ns simulations.

It is well established that the dynamic motion of proteins plays an important functional role, and that the adaptation of a protein molecule to its environment requires optimization of internal non-covalent interactions and protein-solvent interactions. Serine proteinases in general, and trypsin in particular has been used as a model system in exploring possible structural features for cold adaptation. In this study, a 500 p.s. and a 1200 p.s. molecular dynamics (MD) simulation at 300 K of both anionic salmon trypsin and cationic bovine trypsin are analyzed in terms of molecular flexibility, internal non-covalent interactions and protein-solvent interactions. The present MD simulations do not indicate any increased flexibility of the cold adapted enzyme on an overall basis. However, the apparent higher flexibility and deformability of the active site of anionic salmon trypsin may lower the activation energy for ligand binding and for catalysis, and might be a reason for the increased binding affinity and catalytic efficiency compared to cationic bovine trypsin.

Animals↗

PubMed1998

The crystal structure of anionic salmon trypsin in complex with bovine pancreatic trypsin inhibitor.

The complex formed between anionic salmon trypsin (ST) and bovine pancreatic trypsin inhibitor (BPTI) has been crystallised, and the X-ray structure has been solved using the molecular replacement method. The crystals are hexagonal and belong to space group P6(1)22 with lattice parameters of a = b = 83.12 A and c = 222.15 A. Data have been collected to 2.1 A and the structure has been refined to a crystallographic R-factor of 20.6%. Catalysis by salmon trypsin is distinguished by a Km value 20-fold lower than that for mammalian trypsins, and a k(cat) twice as high. The present ST-BPTI complex serves as a model for the Michaelis-Menten complex, and has been compared with corresponding bovine and rat trypsin (RT) complexes. The binding of BPTI to salmon trypsin is characterised by stronger primary interactions in the active site, and a somewhat looser secondary binding.

Animals↗

PubMed1998

Structure of a non-psychrophilic trypsin from a cold-adapted fish species.

The crystal structure of cationic trypsin (CST) from the Atlantic salmon (Salmo salar) has been refined at 1.70 A resolution. The crystals are orthorhombic, belong to space group P212121, with lattice parameters a = 65.91, b = 83.11 and c = 154.79 A, and comprise four molecules per asymmetric unit. The structure was solved by molecular replacement with AMoRe and refined with X-PLOR to an R value of 17.4% and Rfree of 21.5% for reflections |F| > 3sigmaF between 8.0 and 1.7 A resolution. The four non-crystallographic symmetry (NCS) related molecules in the asymmetric unit display r.m.s. deviations in the range 0.31-0.74 A for main-chain atoms, with the largest differences confined to two loops. One of these is the calcium-binding loop where the electron-density indicates a calcium ion for only one of the four molecules. In order to find structural rationalizations for the observed difference in thermostability and catalytic efficiency of CST, anionic salmon trypsin (AST) and bovine trypsin (BT), the three structures have been extensively compared. The largest deviations for the superimposed structures occur in the surface loops and particularly in the so-called 'autolysis loop'. Both the salmon enzymes possess a high methionine content, lower overall hydrophobicity and enhanced surface hydrophilicity, compared with BT. These properties have so far been correlated to cold-adaptation features, while in this work it is shown that the non-psychrophilic cationic salmon trypsin shares these features with the psychrophilic anionic salmon trypsin.

Adaptation, Physiological↗

PubMed1998

Purification and characterization of pancreatic elastase from North Atlantic salmon (Salmo salar).

An elastase I-like enzyme was purified to homogeneity from the pyloric caeca of North Atlantic salmon (Salmo salar) and compared with porcine elastase I. The molecular weight and isoelectric point were estimated to be 27 kDa and over 9.3, respectively. The pH optimum was between 8.0 and 9.5, and the enzyme was unstable at pH values below 4. Kinetic properties examined using Suc-(Ala)3-p-nitroanilide showed that the catalytic efficiency of salmon elastase was about 2.5 times higher than that of porcine elastase. Furthermore, the salmon enzyme was less stable at lower pH values and temperatures than the porcine enzyme. The preference for amino acids at the primary binding site was found to be different from that of the porcine elastase. The salmon elastase binding pocket seems to prefer more branched aliphatic residues than the porcine elastase.

Animals↗

PubMed1995

Structure of native pancreatic elastase from North Atlantic salmon at 1.61 A resolution.

The crystal structure of native salmon pancreatic elastase (SPE) has been solved by molecular-replacement methods, and refined by conventional conjugate-gradient methods and simulated-annealing techniques. The final R value is 17.2% for 21 389 reflections between 8.0 and 1.61 A, and the corresponding free R value is 23.9%. The overall tertiary structure of SPE is remarkably similar to that of porcine pancreatic elastase I (PPE), to which it shows about 67% sequence identity. The primary structure of SPE is determined from the electron-density maps, and only about 15 side chains are somewhat uncertain. Interesting differences between SPE and PPE, are one sequence deletion assigned to position 186, the residue 192 at the entrance of the specificity pocket is substituted from a Gln in PPE to Asn in SPE, and one of the calcium ligands is different. Furthermore, electron density is missing in SPE for the last three residues of the C-terminal helix. A comparison of the present amino-acid sequence of SPE with other sequences available indicates that SPE belongs to the class 1 pancreatic elastases.

Journal Article↗

PubMed1995

Structure of anionic salmon trypsin in a second crystal form.

Anionic salmon trypsin in a second crystal form (ST-IIB) has been refined at 1.83 A, resolution. The crystals are orthorhombic and belong to space group P2(1)2(1)2 with lattice parameters a = 77.09, b = 82.33 and c = 31.16 A. The present structure has been compared to salmon trypsin as it appears in a previously reported crystal form (ST-IIA) with cell dimensions a = 61.95, b = 84.33 and c = 39.11 A [Smalås & Hordvik (1993). Acta Cryst. D49, 318-330]. The presence of a sulfate group involved in several hydrogen bonds to active-site residues, and the location of an additional benzamidine site in the crystal lattice, are the most striking differences between the present and the previous structure. Superposition of main-chain atoms in the two structures give an overall r.m.s. difference of 0.26 A, with the main differences located to areas with different molecular packing. The overall coordinate error is estimated to be between 0.20 and 0.25 A, by the method of Luzzati.

Journal Article↗