Dynamic properties of the active site of azurin studied by the temperature dependence of the optical spectrum

Summary We report the optical absorption spectra of azurin (Pseudomonas aeruginosa) in the temperature range 290-20 K. The samples used are protein aqueous solutions containing 65% (by Vol.) glycerol as cryoprotectant. The measured spectra are deconvoluted in gaussian components and the temperature dependence of the zeroth, first and second moment of the observed bands is analyzed using the harmonic Franck-Condon approximation for the coupling between electronic transitions and nuclear vibrations. The analysis provides information on the stereodynamic properties of the active site of this protein. The possible functional relevance of these results is also suggested.     

Investigation of the active site of Escherichia coli beta-D-galactosidase by photoaffinity labelling.

3,7-Anhydro-2-azi-1,2-dideoxy-D-glycero-L-manno-(8-3H)octitol++ + (1a) and 3-azibutyl 1-thio-beta-D-(6-3H)galactopyranoside (2a) were synthesised from the unlabelled compounds by reaction with galactose oxidase, then reduction with sodium borotritide. Whereas 1a was an efficient photoaffinity reagent for the beta-D-galactosidase from E. coli, 2a was ineffective. Three 3H-labelled peptides were isolated after digestion of the labelled enzyme with trypsin, one of which was an octapeptide (Trp 158 to Ser 165), which is remote from the segments detected as part of the active site of the enzyme.     

Specific labelling of the active site of T7 RNA polymerase.

We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Structural studies of staphylococcal protease. I. Spin labelling of the active site and a comparison with other proteases.

Staphylococcus aureus protease has been spin-labelled at the active-site serine residue with the monocyclic-phosphorus spin label (MSL), 1-oxyl-2,2,6,6-tetramethyl-4-peperi-dinylethylphosphorofluoridate. The electron paramagnetic resonance (E.P.R.) sbectra of the protease in different buffers at various pH's have been analyzed and compared with those of trypsin, subtilisin BPN', and alpha-chymotrypsin under identical conditions. In a given buffer, the shape of E.P.R. signals of spin-labelled staphylococcal protease is unaffected by pH changes except below pH 4.0, at which a gradual loss of conformational integrity of the active site occurs. In bicarbonate buffer and particularly in acetate buffer, the mobility of the label is much more restricted than in phosphate buffer or in potassium chloride solution. The implications of this finding are discussed in terms of a model whereby the label is able to orient towards two different but adjacent regions of the active site. The relative population of the label in each of these orientations is believed to be buffer-dependent. An attempt to correlate the shape of the te.p.r. signals with the pH values of maximal proteolytic avtivity of the enzyme is also presented. These results show that to obtain meaningful information from a comparative spin label study of the geometry of the active site of serine proteases, particular care should be exercised to assure that the different proteases experience identical conditions of pH, buffer, and temperature.     

Structural rearrangements in the active site of smooth-muscle myosin

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.     

Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor

A 1.9 A resolution X-ray structure of the apo-form of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereo-specific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 A. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Structural characterization of the ADAM 16 disintegrin loop active site

ADAM's have various roles in intercellular adhesion and are thought to function by binding integrins through a 13 amino acid motif called the disintegrin loop. Xenopus laevis sperm express the protein ADAM 16, and peptides with the sequence of its disintegrin loop cause downstream events in eggs that require a rise in intracellular calcium similar to that occurring at fertilization. We characterized the portion of the ADAM 16 disintegrin loop responsible for causing egg activation. A peptide based on the C-terminal half of the motif, which includes a known integrin-binding sequence, is a partial agonist of calcium release. A peptide with the N-terminal sequence of the motif activates eggs in a manner virtually identical to the full-length peptide but lacks a recognized integrin-binding sequence. None of these peptides alter the permeability or fluidity of liposomes made from membrane lipids of X. laevis eggs. This result reflects the fact that the peptides do not cause calcium to leak across the egg membrane and indirectly provides evidence that they act through a receptor on the egg surface. The infrared spectrum of the full-length peptide has a strong absorption peak corresponding to a beta-turn. We predict this structure occurs at the N-terminal sequence MPKT. A rearranged peptide lacking any turns fails to activate eggs. These results provide the first structural information about the active site of an ADAM disintegrin loop. We interpret these results in terms of active site sequences from other ADAM's and the role of integrins during fertilization.     

Affinity labelling of the active site of brain phosphatidylinositol 4-kinase with 5'-fluorosulphonylbenzoyl-adenosine.

5'-p-Fluorosulphonylbenzoyl-adenosine (FSO2BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4-kinase (PtdIns 4-kinase). The incubation of PtdIns 4-kinase with concentrations of FSO2BzAdo as low as 50 microM resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50 microM FSO2BzAdo). The kinetics of inactivation of PtdIns 4-kinase by FSO2BzAdo suggest a two-step mechanism, in which a rapid reversible binding of FSO2BzAdo to the enzyme is followed by a covalent sulphonation step. The first-order rate constant (k2) for the inactivation of PtdIns 4-kinase was calculated to be 0.063 min-1, and the steady-state constant of inactivation (Ki) to be 200 microM. Preincubation of the enzyme with either ATP plus Mg2+, or PtdIns alone, prior to addition of FSO2BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO2BzAdo binds within the active site PtdIns 4-kinase. Moreover, since ATP plus Mg2+ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4-kinase by FSO2BzAdo is within the ATP-binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5'-phosphate and tetranitromethane, are consistent with a catalytically-essential lysine being present within the ATP-binding site of PtdIns 4-kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4-kinase by FSO2BzAdo may be due to the labelling of this lysine residue.     

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.     

Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis

Several amino acids which are conserved in cation-pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H+-ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild-type ATPase is only expressed in galactose medium. A series of mutants with decreasing levels of activity demonstrates that the ATPase is rate-limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5'-isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231----Gly, Cys376----Leu, Lys379----Gln and Asp634----Asn; (c) the mutation Ser234----Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730----Asn, which affects a polar residue conserved in hydrophobic stretches of H+-ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.     

Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling

The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin-labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed.     

Chromosome-encoded beta-lactamases of Citrobacter diversus. Interaction with beta-iodopenicillanate and labelling of the active site.

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.     

Structural study on the active site of the collagenase from Hypoderma lineatum

The collagenase from the larvae Hypoderma lineatum is a serine proteinase sequentially related to the trypsin family. The tryptic peptide containing the serine residue of the active site, labelled with [3H] diisopropylfluorophosphate was isolated and determined to be Ser-Pro-Cys-Phe-Gly-Asp-Ser-Gly-Gly-Pro-(Phe-Ser)-Lys. It is highly conservative with respect to the corresponding peptide in other serine proteinases related to trypsin.     

Proton transfer to a charged dye bound to the alpha-chymotrypsin active site studied by laser photolysis.

Pulsed laser photolysis has been used to study the very rapid relaxation of the complex of alpha-chymotrypsin (EC 3.4.21.1) with the coloured inhibitor Biebrich Scarlet. The light absorption causes the dissociation of the proton in the dye naphthol ring and we are able to follow the recombination process under conditions of different ionic strength and pH. The recombination is markedly influenced by the pH around pH 7. The data suggest the existence of relevant interactions in the active site area between the hydrophobic binding site and the proton relay system of the enzyme.     

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an α-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.     

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an alpha-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.