Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies.

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely.

Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy.

Pressure denaturation of Escherichia coli ribonuclease HI (RNase HI) was studied by Fourier transform infrared (FTIR) and two-dimensional NMR spectroscopy at pD* 3.0 and 25 degrees C. A reversible transition in the pressure range of 0.

A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1. 8 A resolution.

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A.

Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses.

The relationship between the structural stability and the internal motions of proteins was investigated through measurements of 15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His), which has a higher melting temperature by 20.2 degreesC. For most of the residues, the generalized order parameters (S2) obtained from 15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast H-D exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions.

Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core.

Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala52 and Val74, resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu.

Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter.

Lipocalin-type prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D2 in the central nervous system and the genital organs and is secreted into the cerebrospinal fluid and the seminal plasma as beta-trace. Here we analyzed retinoids binding of the enzyme by monitoring the fluorescence quenching of an intrinsic tryptophan residue, and appearance of circular dichroism around 330 nm, and a red shift of the UV absorption spectra of retinoids. We found that the enzyme binds all-trans- or 9-cis-retinoic acid and all-trans- or 13-cis-retinal, but not all-trans-retinol, with affinities (Kd of 70-80 nM) sufficient for function as a retinoid transporter.

Solution structure of an extracellular domain containing the WSxWS motif of the granulocyte colony-stimulating factor receptor and its interaction with ligand.

We have determined the NMR structure of a ligand-binding domain of the granulocyte colony-stimulating factor (G-CSF) receptor, containing the highly conserved WSxWS motif. The domain consists of seven beta-strands with the fibronectin type III-like topology seen in several cytokine receptors. Comparisons between the spectra of the 15N-labelled domain with and without G-CSF indicate that the major ligand-recognition site is on the FG loop just upstream of the WSxWS sequence, and not on the BC loop which is mainly used in the growth hormone system.

Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study.

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

Background: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions.

Crystallization and preliminary X-ray diffraction studies of a novel killer toxin from a halotolerant yeast Pichia farinosa.

A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations. Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.

Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48.

Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions.

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I.

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252.

Integrin-specific tissue-type plasminogen activator engineered by introduction of the Arg-Gly-Asp sequence.

To tailor tissue-type plasminogen activator (tPA) to possess an affinity for the integrins, several mutants were constructed by introducing the Arg-Gly-Asp (RGD) sequene into the tPA molecule. These mutants were expressed in COS-1 cells and partially purified by lysine-Sepharose chromatography. The RGD-dependent binding of the mutants to platelet integrin, integrin alpha IIb beta 3, was evaluated by subtracting the nonspecific binding in the presence of 10 mM EDTA (or 1 mg/ml GRGDSP).

Two-dimensional crystals of the Kdp-ATPase of Escherichia coli.

A variant form of the Kdp-ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism. Membranes from overproducing cells were prepared with an inside-out orientation. Incubation of the membranes on ice for 1-2 weeks in the presence of sodium vanadate resulted in the formation of two-dimensional crystals of the Kdp-ATPase.

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.

Three mutants of Escherichia coli ribonuclease HI, in which an invariant acidic residue Asp134 was replaced, were crystallized, and their three-dimensional structures were determined by X-ray crystallography. The D134A mutant is completely inactive, whereas the other two mutants, D134H and D134N, retain 59 and 90% activities relative to the wild-type, respectively. The overall structures of these three mutant proteins are identical with that of the wild-type enzyme, except for local conformational changes of the flexible loops.

The 1.8-A X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding.

The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E.

A role of PDI in the reductive cleavage of mixed disulfides.

We previously reported that protein disulfide isomerase (PDI) can dissociate the glutathione molecule in vitro from the mutant human lysozyme (hLZM) C77A-a, which is modified with glutathione at Cys95; however, it seems structurally difficult for PDI to attack either the disulfide bond or the side chain of the cysteine residue of a mixed disulfide. To investigate the function of PDI, we introduced several glutathione and cysteine derivatives at Cys95, instead of the glutathione of C77A-a. Using thiol compounds modified by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), we could easily modify the free thiol group of C77A-b (C77A with no glutathionylation), without denaturation.

[Catalytic antibodies: generation of a new class of protein catalysts exploiting the immune system].

Since the diversity of the immune response permits antibodies to be generated against virtually any substance, catalytic antibodies have the potential to provide catalysts for many chemical transformations. Such catalysts could be tailored by immunization with rationally designed transition state analogs, which is the main attraction of the catalytic antibody research field. However, when mice are immunized with a hapten conjugated to a carrier protein, a few, and occasionally several, of the dozens of antibodies that bind the hapten are characterized as catalytic.

Tailoring echistatin to possess higher affinity for integrin alpha(IIb)beta(3).

A mutant of echistatin, a disintegrin with a high affinity for the integrins, was constructed by substituting CRGDC for ARGDD in the Arg-Gly-Asp (RGD) region. The mutant was chemically synthesized, subjected to a folding process with air oxidation, and purified by reverse-phase HPLC. The peptide mapping and mass spectrometric analyses revealed that the two Cys residues introduced in the mutant are linked to each other, without any effect on the mode of the four disulfide bonds present in native echistatin, as expected.

Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli. The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution.

Crystallization and preliminary X-ray analysis of the primary receptor (PotD) of the polyamine transport system in Escherichia coli.

The primary receptor (potD, M(r) = 39 000) of the polyamine transport system in Escherichia coli has been crystallized by the vapor-diffusion method. Two crystal forms were obtained in the presence of spermidine, and were examined by X-ray analysis. Form I crystals, which diffract to 2.

Synthetic collagen-like domain derived from the macrophage scavenger receptor binds acetylated low-density lipoprotein in vitro.

The bovine macrophage scavenger receptor is a 70 kDa membrane protein that is trimerized on the macrophage cell surface. The receptor binds modified low-density lipoproteins (LDL). The core binding site is located within 22 residues at the C-terminus of the collagen-like domain of the receptor.

Roles of electrostatic interaction in proteins.

More than 60 years after the analyses by Linderstrom-Lang and Kirkwood of their hypothetical 'protein' structures, we have now a plethora of experimental evidence and computational estimates of the electrostatic forces in proteins, with very many protein 3D structures at atomic resolution. In the mean time, there were in the beginning, many arguments and suggestions about the roles of electrostatics, mainly from empirical findings and tendencies. A few experimental results indicated that the electrostatic contribution is of the order of several kcal/mol, which was theoretically difficult to reproduce correctly, because a large opposing reaction field should be subtracted from a large, direct Coulombic field.

High helicity of peptide fragments corresponding to beta-strand regions of beta-lactoglobulin observed by 2D-NMR spectroscopy.

Background: Whereas protein fragments, when they are structured, adopt conformations similar to that found in the native state, the high helical propensity of beta-lactoglobulin, a predominantly beta-sheet protein, suggested that the fragments of beta-lactoglobulin can assume the non-native helical conformation. In order to assess this possibility, we synthesized four 17-18-residue peptides corresponding to three beta-strand regions and one helical region (as a control) of beta-lactoglobulin and examined their conformation.

Results: We observed residual helicities of up to 17% in water, by far-UV CD, for all four peptide fragments.