Basic folded and low-populated locally disordered conformers of SUMO-2 characterized by NMR spectroscopy at varying pressures

SUMO proteins, a group of post-translational ubiquitin-like modifiers, have target enzymes (E1 and E2) like other ubiquitin-like modifiers, e.g., ubiquitin and NEDD8, but their physiological roles are quite different. In an effort to determine the characteristic molecular design of ubiquitin-like modifiers, we have investigated the structure of human SUMO-2 in solution not only in its basic folded state but also in its higher-energy state by utilizing standard and variable-pressure NMR spectroscopy, respectively. We have determined average coordinates of the basic folded conformer at ambient pressure, which gives a backbone structure almost identical with those of ubiquitin and NEDD8. We have further investigated conformational fluctuations in a wide conformational space using variable-pressure NMR spectroscopy in the range of 30-3 kbar, by which we find a low-populated ( approximately 2.5%) alternative conformer preferentially disordered in the enzyme-binding segment. The alternative conformer is structurally very close to but markedly different in equilibrium population from those for ubiquitin and NEDD8. These results support our notion that post-translational ubiquitin-like modifiers are evolutionarily designed for function both structurally and thermodynamically in their low-populated, high-energy conformers rather than in their basic folded conformers.     

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.     

The pressure-temperature free energy-landscape of staphylococcal nuclease monitored by (1)H NMR

The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing (1)H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % (2)H(2)O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 degrees C-35 degrees C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Deltabeta), the volume change at 1 bar (DeltaV degrees ) and the expansivity change (Deltaalpha) upon unfolding among other thermodynamic parameters: Deltabeta=0.02(+/-0.003) ml mol(-1) bar(-1); Deltaalpha=1.33(+/-0.2) ml mol(-1) K(-1); DeltaV degrees =-41.9(+/-6. 3) ml mol(-1) (at 24 degrees C); DeltaG degrees =13.18(+/-2) kJ mol(-1) (at 24 degrees C); DeltaC(p)=13.12(+/-2) kJ mol(-1) K(-1); DeltaS degrees =0.32(+/-0.05) kJ mol(-1) K(-1 )(at 24 degrees C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Deltabeta and Deltaalpha values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.     

Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate

The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native alpha-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI) (0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU) (0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins, belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies.     

No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.     

Mechanical unfolding pathway and origin of mechanical stability of proteins of ubiquitin family: An investigation by steered molecular dynamics simulation

Like the muscle protein Titin, proteins of the ubiquitin family exhibit a parallel strand arrangement, but otherwise having a distinctly different fold and not involved in an obvious load‐bearing function, exhibit high resistance to mechanical unfolding. We have applied all‐atom molecular dynamics simulation technique in implicit solvent to present a deep insight into the force‐induced unfolding pathway of three proteins—ubiquitin, NEDD8, and SUMO‐2—all having almost similar structural features. Two intermediates evolve in the unfolding pathway of each of the three proteins. The first intermediate, which has already been identified in case of ubiquitin by earlier simulation results, is similar for ubiquitin and NEDD8, but different in SUMO‐2. We have found a new intermediate with β3–β4 hairpin and some residual α‐helical character; and this intermediate is common for all the three proteins. Thus, proteins of the ubiquitin family pass through a well‐defined conformation in their force‐induced unfolding pathway. Reason behind the higher mechanical stability of the proteins with parallel strand structures like Titin has also been identified. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.     

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.     

Kinetic barriers to the folding of horse cytochrome C in the reduced state

To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor.     

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.     

The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.     

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

Apo-azurin folds via an intermediate that resembles the molten-globule

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.     

Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21

Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: iron cofactors dissociate after polypeptide unfolding

Here we report the conformational stability of homodimeric desulfoferrodoxin (dfx) from Desulfovibrio desulfuricans (ATCC 27774). The dimer is formed by two dfx monomers linked through beta-strand interactions in two domains; in addition, each monomer contains two different iron centers: one Fe-(S-Cys)(4) center and one Fe-[S-Cys+(N-His)(4)] center. The dissociation constant for dfx was determined to be 1 microM (DeltaG = 34 kJ/mol of dimer) from the concentration dependence of aromatic residue emission. Upon addition of the chemical denaturant guanidine hydrochloride (GuHCl) to dfx, a reversible fluorescence change occurred at 2-3 M GuHCl. This transition was dependent upon protein concentration, in accord with a dimer to monomer reaction [DeltaG(H(2)O) = 46 kJ/mol of dimer]. The secondary structure did not disappear, according to far-UV circular dichroism (CD), until 6 M GuHCl was added; this transition was reversible (for incubation times of < 1 h) and independent of dfx concentration [DeltaG(H(2)O) = 50 kJ/mol of monomer]. Thus, dfx equilibrium unfolding is at least three-state, involving a monomeric intermediate with native-like secondary structure. Only after complete polypeptide unfolding (and incubation times of > 1 h) did the iron centers dissociate, as monitored by disappearance of ligand-to-metal charge transfer absorption, fluorescence of an iron indicator, and reactivity of cysteines to Ellman's reagent. Iron dissociation took place over several hours and resulted in an irreversibly denatured dfx. It appears as if the presence of the iron centers, the amino acid composition, and, to a lesser extent, the dimeric structure are factors that aid in facilitating dfx's unusually high thermodynamic stability for a mesophilic protein.