Glycerol-slaved 1H-1H NMR cross-relaxation in quasi-native lysozyme.

1H-1H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1H-1H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol Ds with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (Ds âˆ¼ Î·-κ, κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation.

The food and pharmaceutical additive benzoic acid induces amyloid fibrillation of an intrinsically disordered protein.

Benzoic acid (BA) is a microbe-inhibiting flavoring agent used extensively as an additive in foods, pharmaceuticals, and hygiene and cosmetic products. The level of BA in foodstuffs prescribed by world bodies and governmental agencies is assumed to be safe so as to prevent adverse health effects. The safety level of BA is however controversial, and whether different conditions of its use would be generally regarded as safe (GRAS) has been rarely determined. In the quest of how food additives affect the structure and conformation of proteins, this study evaluates the interaction of BA with an intrinsically disordered protein (IDP) at pH 4.2 that matches the pH conditions applicable for the commercial use of benzoate preservatives, and examines its structural transformation by NMR, fluorescence, and high-resolution microscopy. The interaction with BA transforms the protein to a denatured aggregated mesophase that undergoes reconfiguration to yield rigid amyloid fibrils. Significantly, fibrils are observed even with 0.1 mM BA while the recommended level of its use as a preservative is in the 0.4-8 mM range. The discussion refrains from safety comments with no projection of the BA level that could be GRAS.

A three-state mechanism for trifluoroethanol denaturation of an intrinsically disordered protein (IDP).

Relating the amino acid composition and sequence to chain folding and binding preferences of intrinsically disordered proteins (IDPs) has emerged as a huge challenge. While globular proteins have respective 3D structures that are unique to their individual functions, IDPs violate this structure-function paradigm because rather than having a well-defined structure an ensemble of rapidly interconverting disordered structures characterize an IDP. This work measures 2,2,2-trifluoroethanol (TFE)-induced equilibrium transitions of an IDP called AtPP16-1 (Arabidopsis thaliana phloem protein type 16-1) by using fluorescence, circular dichroism, infrared and nuclear magnetic resonance (NMR) methods at pH 4, 298 K. Low TFE reversibly removes the tertiary structure to produce an ensemble of obligate intermediate ($\mathrm{I}$) retaining the native-state ($\mathrm{N}$) secondary structure. The intermediate $\mathrm{I}$ is preceded by a non-obligate tryptophan-specific intermediate ${\mathrm{I}}_{\mathrm{w}}$ whose population is detectable for AtPP16-1 specifically. Accumulation of such non-obligate intermediates is discriminated according to the sequence composition of the protein. In all cases, however, a tertiary structure-unfolded general obligate intermediate $\mathrm{I}$ is indispensable. The $\mathrm{I}$ ensemble has higher helical propensity conducive to the acquisition of an exceedingly large level of α-helices by a reversible denaturation transition of $\mathrm{I}$ to the denatured state $\mathrm{D}$ as the TFE level is increased. Strikingly, it is the same $\mathrm{N}\rightleftharpoons \mathrm{I}\rightleftharpoons \mathrm{D}$ scheme typifying the TFE transitions of globular proteins. The high-energy state $\mathrm{I}$ characterized by increased helical propensity is called a universal intermediate encountered in both genera of globular and disordered proteins. Neither $\mathrm{I}$ nor $\mathrm{D}$ strictly show molten globule (MG)-like properties, dismissing the belief that TFE promotes MGs.

Mechanical Unfolding and Amorphous Aggregation of Protein in a Very Low DC Field.

A common theme for the effect of electric field on the structure and conformation of proteins is lacking due to a myriad of conflicting reports emerging from different protein systems subjected to different frequencies and strengths of the field (0.8 -108 V cm-1), which may be pulsed for a few nano- to microseconds or applied continuously up to several hours. It is however necessary to find a common theme because of the increasing use of electric field not only to understand Stark-like electro-optic effects in large molecules but also in food processing technology, and perhaps in the disruption of amyloid bodies in Alzheimer's condition. This study finds an optimized condition of 1.3 V cm-1 DC field, in which the electrophoretic mobility is ∼1.2 mm h-1, and systematically shows electrophoretic, electrochemical, and unfolding effects at different levels of cytochrome c structure within ∼90 min of turning the field on. Interestingly, the protein undergoes amorphous aggregation concomitant with a high degree of denaturation. In support of this suggestion, data for myoglobin and trypsin are also presented. Effort has been made to separate out the chemical and physical effects of the electric field.

A surprisingly simple three-state generic process for reversible protein denaturation by trifluoroethanol.

Despite the rich knowledge of the influence of 2,2,2-trifluoroethanol (TFE) on the structure and conformation of peptides and proteins, the mode(s) of TFE-protein interactions and the mechanism by which TFE reversibly denatures a globular protein remain elusive. This study systematically examines TFE-induced equilibrium transition curves for six paradigmatic globular proteins by using basic fluorescence and circular dichroism measurements under neutral pH conditions. The results are remarkably simple. Low TFE invariably unfolds the tertiary structure of all proteins to produce the obligate intermediate (I) which retains nearly all of native-state secondary structure, but enables the formation of extra α-helices as the level of TFE is raised higher. Inspection of the transitions at once reveals that the tertiary structure unfolding is always a distinct process, necessitating the inclusion of at least one obligate intermediate in the TFE-induced protein denaturation. It appears that the intermediate in the minimal unfolding mechanism N⇌I⇌D somehow acquires higher α-helical propensity to generate α-helices in excess of that in the native state to produce the denatured state (D), also called the TFE state. The low TFE-populated intermediate I may be called a universal intermediate by virtue of its α-helical propensity. Contrary to many earlier suggestions, this study dismisses molten globule (MG)-like attribute of I or D.

Negative Thermal Expansion and Disorder-to-Order Collapse of an Intrinsically Disordered Protein under Marginally Denaturing Conditions.

Recent work with intrinsically disordered proteins (IDPs) has projected a myriad of their survival instincts based mainly on the total charge content, the abundance of polar residues, and the paucity of hydrophobic amino acids. This work uses a plant IDP AtPP16-1 (Arabidopsis thaliana phloem protein class 16-1), whose solution NMR structure was determined by us recently, to show legitimate negative thermal expansion (NTE) of the native state. The thermal expansion continues to be negative even as the tertiary structure is perturbed by ultralow levels of urea up to 0.4 M. The NTE of these subdenatured states is called apparent NTE because they are prone to undergo conformational changes with temperature. Hydrodynamic shrinkage of the NTE IDP is also observed by dynamic light scattering (DLS) and NMR-measured global rotational correlation time (τc). The protein with denatured tertiary structure but otherwise preserved native-state secondary structure collapses to a dynamically rigid state. The data are mainly based on thermal coefficients of chemical shift and nuclear relaxation measured by heteronuclear NMR. The hydrodynamic shrinkage and collapse under marginally varying solvent compositions that may arise from unstable tertiary structure and dynamic disorder of chain segments across the backbone could be a generic property of IDPs.

Quasi-native transition and self-diffusion of proteins in water-glycerol mixture.

An orderly investigation of the levels of secondary and tertiary structures, kinetics of tertiary structural changes, and self diffusion coefficient of lysozyme and cytochrome c in the 0-70% (weight/volume) range of glycerol is reported. While secondary structural propensity of both proteins is larger in glycerol, results for tertiary structure and translational diffusion coefficient with increasing glycerol provide two contrasting depictions - lysozyme becomes increasingly compact, plausibly due to disulfide bridge constraints, but cytochrome c expands and loses the tertiary structure. The chain expansion and contraction corresponding to loss and reformation of tertiary structure of cytochrome c are ultrafast that occur in the submillisecond bin. Changes in protein conformation appear in as little as 2% glycerol, and the results suggest that glycerol does not unfold the protein but reversibly destabilizes to quasi-native state(s). These observations make one ponder whether results of studies on protein dynamics, relaxation, and conformational substates reported in literature can be associated with native-state properties.

Complete observation of all structural, conformational, and fibrillation transitions of monomeric globular proteins at submicellar sodium dodecyl sulfate concentrations.

Although considerable information is available regarding protein-sodium dodecyl sulfate (SDS) interactions, it is still unclear as to how much SDS is needed to denature proteins. The role of protein charge and micellar surfactant concentration on amyloid fibrillation is also unclear. This study reports on equilibrium measurements of SDS interaction with six model proteins and analyzes the results to obtain a general understanding of conformational breakdown, reorganization and restructuring of secondary structure, and entry into the amyloid fibrillar state. Significantly, all of these responses are entirely resolved at much lower than the critical micellar concentration (CMC) of SDS. Electrostatic interaction of the dodecyl sulfate anion (DS- ) with positive surface potential on the protein can completely unfold both secondary and tertiary structures, which is followed by protein chain restructuration to α-helices. All SDS-denatured proteins contain more α-helices than the corresponding native state. SDS interaction stochastically drives proteins to the aggregated fibrillar state.

Amyloid fibrillation of an intrinsically disordered plant phloem protein AtPP16-1 under acidic condition.

Arabidopsis thaliana Phloem Protein 16-1 (AtPP16-1) is a 156-residue intrinsically disordered nucleic acid binding protein which is putatively involved in long-distance systemic transport of RNA to budding regions in plants. Dimerization or oligomerization of the protein at pH higher than about 4.1 leaves no apolar surface exposed for interaction with the dye 8-Anilinonaphthalene-1-sulfonate (ANS). The most stable monomeric state is found near pH 4 where the structure of the protein is determined to have three short ß-strands and a single α-helix. By surveying the pH-dependent propensity of fibrillation we find the protein enters the amyloidogenic state at pH 2, 60 °C. The reaction product is not amorphous aggregate, but simple amyloid fibrils with sparse or no branching. The mean diameters of the fibril population scaled from AFM images are 13.2 and 21.2 nm for precursor aggregates (PA) and proto- or elongated fibrils, respectively. These values are somewhat larger than the fibril diameters generally cited, and the reason could be larger lateral association for both PA and protofibrils. The protein AtPP16-1 is strictly pH-selective in terms of its structure and stability, and the solution structure is known at pH 4. Under the conditions of pH 2 used here for fibrillation, the protein retains substantial secondary structure. Even if the pH and temperature conditions used for fibrillation are hardly physiological, there is a finite possibility that some aggregation of AtPP16-1 would occur in vivo, as the case of transglutaminase aggregates in the chloroplast of transplastomic plants, for example. The pH related problem has been discussed in detail, but the questions emanating are: do phloem proteins fibrillate in vivo, and if so what implication fibrillation has for plant physiology?

Solution NMR Structure and Backbone Dynamics of Partially Disordered Arabidopsis thaliana Phloem Protein 16-1, a Putative mRNA Transporter.

Although RNA-binding proteins in plant phloem are believed to perform long-distance systemic transport of RNA in the phloem conduit, the structure of none of them is known. Arabidopsis thaliana phloem protein 16-1 (AtPP16-1) is such a putative mRNA transporter whose structure and backbone dynamics have been studied at pH 4.1 and 25 °C by high-resolution nuclear magnetic resonance spectroscopy. Results obtained using basic optical spectroscopic tools show that the protein is unstable with little secondary structure near the physiological pH of the phloem sap. Fluorescence-monitored titrations reveal that AtPP16-1 binds not only A. thaliana RNA (Kdiss ∼ 67 nM) but also sheared DNA and model dodecamer DNA, though the affinity for DNA is ∼15-fold lower. In the solution structure of the protein, secondary structural elements are formed by residues 3-9 (ß1), 56-62 (ß2), 133-135 (ß3), and 96-110 (α-helix). Most of the rest of the chain segments are disordered. The N-terminally disordered regions (residues 10-55) form a small lobe, which conjoins the rest of the molecule via a deep and large irregular cleft that could have functional implications. The average order parameter extracted by model-free analysis of 15N relaxation and {1H}-15N heteronuclear NOE data is 0.66, suggesting less restricted backbone motion. The average conformational entropy of the backbone NH vectors is -0.31 cal mol-1 K-1. These results also suggest structural disorder in AtPP16-1.

Dispersion Forces and the Molecular Origin of Internal Friction in Protein.

Internal friction in macromolecules is one of the curious phenomena that control conformational changes and reaction rates. It is held here that dispersion interactions and London-van der Waals forces between nonbonded atoms are major contributors to internal friction. To demonstrate this, the flipping motion of aromatic rings of F10 and Y97 amino acid residues of cytochrome c has been studied in glycerol/water mixtures by cross relaxation-suppressed exchange nuclear magnetic resonance spectroscopy. The ring-flip rate is highly overdamped by glycerol, but this is not due to the effect of protein-solvent interactions on the Brownian dynamics of the protein, because glycerol cannot penetrate into the protein to slow the internal collective motions. Sound velocity in the protein under matching solvent conditions shows that glycerol exerts its effect by rather smothering the protein interior to produce reduced molecular compressibility and root-mean-square volume fluctuation (δVRMS), implying an increased number of dispersion interactions of nonbonded atoms. Hence, δVRMS can be used as a proxy for internal friction. By using the ansatz that internal friction is related to nonbonded interactions by the equation f(n) = f0 + f1n + f2n(2) + ..., where the variable n is the extent of nonbonded interactions with fi coefficients, the barrier to aromatic ring rotation is found to be flat. Also interesting is the appearance of a turnover region in the δVRMS dependence of the ring-flip rate, suggesting anomalous internal diffusion. We conclude that cohesive forces among nonbonded atoms are major contributors to the molecular origin of internal friction.

Viscosity Dependence of Some Protein and Enzyme Reaction Rates: Seventy-Five Years after Kramers.

Kramers rate theory is a milestone in chemical reaction research, but concerns regarding the basic understanding of condensed phase reaction rates of large molecules in viscous milieu persist. Experimental studies of Kramers theory rely on scaling reaction rates with inverse solvent viscosity, which is often equated with the bulk friction coefficient based on simple hydrodynamic relations. Apart from the difficulty of abstraction of the prefactor details from experimental data, it is not clear why the linearity of rate versus inverse viscosity, k ∝ η(-1), deviates widely for many reactions studied. In most cases, the deviation simulates a power law k ∝ η(-n), where the exponent n assumes fractional values. In rate-viscosity studies presented here, results for two reactions, unfolding of cytochrome c and cysteine protease activity of human ribosomal protein S4, show an exceedingly overdamped rate over a wide viscosity range, registering n values up to 2.4. Although the origin of this extraordinary reaction friction is not known at present, the results indicate that the viscosity exponent need not be bound by the 0-1 limit as generally suggested. For the third reaction studied here, thermal dissociation of CO from nativelike cytochrome c, the rate-viscosity behavior can be explained using Grote-Hynes theory of time-dependent friction in conjunction with correlated motions intrinsic to the protein. Analysis of the glycerol viscosity-dependent rate for the CO dissociation reaction in the presence of urea as the second variable shows that the protein stabilizing effect of subdenaturing amounts of urea is not affected by the bulk viscosity. It appears that a myriad of factors as diverse as parameter uncertainty due to the difficulty of knowing the exact reaction friction and both mode and consequences of protein-solvent interaction work in a complex manner to convey as though Kramers rate equation is not absolute.

Free energy landscape of lysozyme: multiple near-native conformational states and rollover in the urea dependence of folding energy.

Deviation from linearity of the equilibrium folding free energy (ΔG) of proteins along the reaction coordinate is scarcely known. Optical spectroscopic observables and NMR-measured average molecular dimensional property of lysozyme with urea at pH 5 reveal that ΔG rolls over from linearity under mild to strongly native-like conditions. The urea dependence of ΔG is graphed in the 0-7 M range of the denaturant by employing a series of guanidine hydrochloride (GdnHCl)-induced equilibrium unfolding transitions, each in the presence of a fixed level of urea. The observed linear dependence of ΔG on urea under denaturing conditions begins to deviate as moderately native-like conditions are approached and eventually rolls over under strongly native-like conditions. This is atypical of the upward curvature in the ΔG vs denaturant plot predicted by the denaturant binding model. On increasing the denaturant concentration from 0 to 5 M, the hydrodynamic radius of lysozyme shrinks by ∼2 Å. We suggest subdenaturing levels of urea affect the population distribution among multiple near-native isoenergetic conformational states so as to promote them sequentially with increments of the denaturant. We use a multiple-state sequential model to show that the keel over of ΔG occurs due to these near-native alternative states in the native ensemble used for defining the unfolding equilibrium constant (KU), which we assume to vary linearly with urea. The results and the model appear to indicate a rugged flat bottom in the free energy landscape wherein population distribution of native-like states is modulated by urea-affected interstate motions.

Preferential water exclusion in protein unfolding.

Association of water with protein plays a central role in the latter's folding, structure acquisition, ligand binding, catalytic reactivity, oligomerization, and crystallization. Because these phenomena are also influenced by the net charge content on the protein, the present study examines the association of water with cytochrome c held at different pH values so as to allow its side chains to ionize to variable extents. Equilibrium unfolding of differently charged cytochrome c molecules in water-methanol binary mixtures, where the alcohol acts as the cosolvent denaturant, was used to quantify the preferential exclusion of water during the unfolding transition. The extent of exclusion was found to be related to the net-charge-dependent molecular expansion of the protein in an alcohol-free aqueous medium. The degree of water exclusion was also found to be linearly related to the observed rate of protein unfolding, where the net charge contents of the initial and final states are the same. The results suggest that side-chain ionization, molecular expansion due to charge repulsion, and hence the loss of tertiary contacts lead to additional water-protein association. Protein unfolding rates appear to be linearly correlated with the effective number of water molecules excluded across the end states of unfolding equilibria.

Expansion and internal friction in unfolded protein chain.

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome c (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction.

Enzyme dimension of the ribosomal protein S4 across plant and animal kingdoms.

BACKGROUND: The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO). METHODS: Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells. RESULTS: Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B, human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic kidney cells, presumably due to the extraribosomal enzyme trait reported. CONCLUSIONS: The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases in the extraribosomal milieu. GENERAL SIGNIFICANCE: The enzyme nature of S4 is relevant toward understanding not only the origin of ribosomal proteins, but also processes in cell biology and diseases.

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.

BACKGROUND: It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. METHODS: Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. RESULTS: The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. CONCLUSION: The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. SIGNIFICANCE: Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.

Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4.

S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α-operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell-free system of rabbit reticulocytes. The translation-blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.

Hydrophobic collapse overrides Coulombic repulsion in ferricytochrome c fibrillation under extremely alkaline condition.

Tuning of both hydrophobic and electrostatic interactions is thought to be important for the initial nucleation and stability of protein aggregates that self-assemble to produce amyloid fibrils. Importance of a critical balance of these two interactions has indeed been determined under various solution conditions of fibrillation, the acidic pH, in particular. To find out if fibrillar protein structures could be obtained under extreme alkaline conditions, cytochrome c was allowed to fibrillate in 0.1 N NaOH at 50 or 60 °C. Fibers do grow in alkali, but the fibrillation process depends little on the ionic strength of the solution. Illustrative fibril morphology readily obtained even in the absence of solvent cations poses the question as to how the severity of electrostatic repulsions is overcome to initiate aggregation. It appears that intermolecular hydrophobic collapse is so overwhelming that electrostatic repulsions are subdued, and the negative charges on protein molecules are relocated in a way conducive to fiber growth. This proposal seems consistent with computer simulation studies indicating central role of hydrophobic interactions. Morphologically, branched fibrils characterized by a wide distribution of diameter are assembled by winding two or more protofibrils. The results should guide selection of model parameters in theoretical studies of fibrillation.

Cysteine protease attribute of eukaryotic ribosomal protein S4.

BACKGROUND: Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow. METHODS: Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac-XEXD↓-AFC (N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR↓-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions. RESULTS: Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K(m) value for the cleavage of Z-FR↓-AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg(2+) and Zn(2+) concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac-LEHD↓-AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z-FR↓-AMC. GENERAL SIGNIFICANCE: The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins.