Balanced Electrostatic and Structural Forces Guide the Large Conformational Change Associated with Maturation of T = 4 Virus

Nudaurelia capensis omega virus has a well-characterized T = 4 capsid that undergoes a pH-dependent large conformational changes (LCC) and associated auto-catalytic cleavage of the subunit. We examined previously the particle size at different pH values and showed that maturation occurred at pH 5.5. We now characterized the LCC with time-resolved small-angle x-ray scattering and showed that there were three kinetic stages initiated with an incremental drop in pH: 1), a rapid (<10 ms) collapse to an incrementally smaller particle; 2), a continuous size reduction over the next 5 s; and 3), a smaller final transition occurring in 2-3 min. Equilibrium measurements similar to those reported previously, but now more precise, showed that the particle dimension between pH 5.5 and 5 requires the autocatalytic cleavage to achieve its final compact size. A balance of electrostatic and structural forces shapes the energy landscape of the LCC with the latter requiring annealing of portions of the subunit. Equilibrium experiments showed that many intermediate states could be populated with a homogeneous ensemble of particles by carefully controlling the pH. A titration curve for the LCC was generated that showed that the virtual pK_a (i.e., the composite of all titratable residues that contribute to the LCC) is 5.8.     

Comparison of the effects of bile acids and GSH on the fluorescence of bound 1-anilino-8-naphthalene sulfonate and the enzymatic activity of cationic and neutral human hepatic GSH S-transferases

Three cationic (C1, C2, A1) and a neutral (N1) glutathione (GSH) S-transferase were purified to homogeneity from human liver, as we have previously reported. GSH had no effect on the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS) bound by transferase C1 and N1, but markedly enhanced the fluorescence with C2 and A1 without changing the affinity for ANS. This effect of GSH was saturable and with C2 was intermediate between A1 and C1. Bile acids inhibited the fluorescence of ANS bound to C1 and C2. GSH in the presence of bile acids further decreased the fluorescence of ANS bound to C1 and increased the fluorescence with C2. Transferase A1 showed decreased fluorescence in the presence of lithocholic acid and increased fluorescence in the presence of cholic acid; both changes were reversed by GSH. Transferase N1 showed increased fluorescence of bound ANS in the presence of various bile acids and this effect was diminished in the presence of GSH. Enzyme activity of the transferase was inhibited by bile acids with the exception of transferase A1. All the proteins bound lithocholic acid. The inhibition of C1 and N1 was greater at pH 6.5 than 7.4 and the order of addition of substrates and inhibitor made no difference.     

On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition

Tripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD-OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non-transported beta-lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD-OprJ-mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD-OprJ susceptible to inhibition by a thiol-reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu-Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.     

Interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and human matrix metalloproteinase 7 (MMP-7) as examined by MMP-7 activity and ANS fluorescence

Human matrix metalloproteinase 7 (MMP-7) is the smallest matrix metalloproteinase. It plays important roles in tumour invasion and metastasis. 8-Anilinonaphthalene 1-sulphonate (ANS) is a fluorescent probe widely used for the analysis of proteins. It emits large fluorescence energy when its anilinonaphthalene group binds with hydrophobic regions of protein. In this study, we analysed the interaction of ANS and MMP-7. At pH 4.5-9.5, ANS inhibited MMP-7 activity in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2). The inhibition was a non-competitive manner and depended on the time for pre-incubation of ANS and MMP-7. At pH 4.5-9.5, the fluorescence of ANS was not changed by the addition of MMP-7. At pH 3.5, MMP-7 lacked activity, and the fluorescence of ANS was increased by the addition of MMP-7. These results suggest that at pH 4.5-9.5, the sulphonic group of ANS binds with MMP-7 through electrostatic interaction, whereas at pH 3.5, the anilinonaphthalene group of ANS binds with MMP-7 through hydrophobic interaction.     

Kinetic intermediates of unfolding of dimeric prostatic phosphatase

Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20 degrees C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 +/- 0.0024 min(-1) . The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 +/- 0.0018 min(-1) and 0.0409 +/- 0.0052 min(-1), respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 +/- 0.014 min(-1) and 0.216 +/- 0.010 min(-1), respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.     

Capsid conformational sampling in HK97 maturation visualized by X-ray crystallography and cryo-EM

Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.     

The minimal structural requirement of concanavalin A that retains its functional aspects

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35 > CHAPS > CTAB > SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.     

Two steps in the transition between the native and acid states of bovine α-lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.     

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

A novel type of phosphofructokinase from plants

A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.     

An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.

The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.     

Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate

Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the folding and stability of the catalytically active oligomeric protein. Unfolding was reversible, as verified by activity measurements and tryptophan fluorescence. The noncoincidence of the unfolding curves obtained by different techniques suggests the occurrence of a multiphasic process.The reaction of hPAP inactivation is accompanied by dissociation of the dimer into two monomers. The midpoint of this transition is at 0.65 M GdnHCl with 4.24+/-0.12 kcalmol(-1) free energy change. Binding of ANS to the inactive phosphatase monomer, especially remarkable in the region from 0.8 to 1.25M GdnHCl, suggests that the hydrophobic probe indicates exposition of the intersubunit hydrophobic surface and a loosening of the monomer's tertiary structure. Strong fluorescence of thiol group derivatives, the products of their reaction with MIANS, appears in a limited range of GdnHCl concentrations (1.2-1.6M). This shows that in the relaxed structure of the intermediate, the reagent is allowed to penetrate into the hydrophobic environment of the partially hidden thiol groups.The equilibrium unfolding reaction of hPAP, as monitored by tryptophan fluorescence, does not depend on the protein concentration and displays a single transition curve with a midpoint at 1.7 M GdnHCl and value of DeltaG(unf)(H(2)O)=3.38+/-0.08 kcalmol(-1) per monomer, a result implying that this transition is related to the conformational change of the earlier dissociated and already inactive subunit of the protein.     

Characterization of a transient covalent adduct formed during dimethylarginine dimethylaminohydrolase catalysis

Dimethylarginine dimethylaminohydrolase (DDAH) regulates the concentrations of human endogenous inhibitors of nitric oxide synthase, N(omega)-methyl-l-arginine (NMMA), and asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA). Pharmacological regulation of nitric oxide synthesis is an important goal, but the catalytic mechanism of DDAH remains largely unexplored. A DDAH from Pseudomonas aeruginosa was cloned, and asymmetrically methylated arginine analogues were shown to be the preferred substrates, with ADMA displaying a slightly higher k(cat)/K(M) value than NMMA. DDAH is similar to members of a larger superfamily of guanidino-modifying enzymes, some of which have been shown to use an S-alkylthiouronium intermediate during catalysis. No covalent intermediates were found to accumulate during steady-state turnover reactions of DDAH with NMMA or ADMA. However, identification of a new substrate with an activated leaving group, S-methyl-l-thiocitrulline (SMTC), enabled acid trapping and ESI-MS characterization of a transient covalent adduct with a mass of 158 +/- 10 Da that accumulates during steady-state turnover. Subsequent trapping, proteolysis, peptide mapping and fragmentation by mass spectrometry, and site-directed mutagenesis demonstrated that this covalent adduct was attached to an active site residue and implicates Cys249 as the catalytic nucleophile required for intermediate formation. The use of covalent catalysis clearly links DDAH to this superfamily of enzymes and suggests that an S-alkylthiouronium intermediate may be a conserved feature in their mechanisms.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Fluorescent probe studies of haptoglobin type 2-1.

Haptoglobin is an alpha2 serum protein that forms an irreversible complex with hemoglobin. The combination between these two macromolecules resembles the binding of an antigen to its antibody except that the complex remains soluble. This investigation was undertaken to determine the nature of the hydrophobic sites on haptoglobin type 2-1. The interaction of 1-anilinonphthalene-8-sulfonate (ANS) with haptoglobin type 2-1 is characterized by a flourescence intensity in solutions containing ANS and haptoglobin as the pH is decreased from 9 to 4. The dissociation constant for the ANS interaction with haptoglobin 2-1 is 5.8 x 10--5 M at pH 7.0, 5.2 X 10--5 M at pH 5.0 AND 30.3 X 10--5 M at pH 4.0. Fmax shows no change in the pH range 6-9 but does show an increase at pH 4.0 when compared to the neutral region.     

Inhibition of yeast cytosine deaminase by 5-bromo-2-pyrimidinone and its covalent hydrate

Yeast cytosine deaminase (EC 3.5.4.1) is inhibited by 5-bromo-2-pyrimidinone. In aqueous solution at neutral pH three forms of this compound (the anion, the parent, and the covalent hydrate) are in equilibrium. Experiments were undertaken in order to determine the relative contributions of these three forms to the observed inhibition. The anion makes little or no contribution. Both the parent and the covalent hydrate inhibit the enzyme, with the Ki for the hydrate being 0.2-0.02 times that of the parent. In the presence of stoichiometric concentrations of the enzyme, the equilibrium between parent and hydrate is displaced towards the hydrate; however, the hydration is not catalyzed by cytosine deaminase.     

Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.     

Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy

We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.     

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris)

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.