Sedimentation Coefficient Research Articles

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

Sedimentation of knotted polymers

We investigate the sedimentation of knotted polymers by means of stochastic rotation dynamics, a molecular dynamics algorithm that takes hydrodynamics fully into account. We show that the sedimentation coefficient s, related to the terminal velocity of the knotted polymers, increases linearly with the average crossing number n(c) of the corresponding ideal knot. This provides direct computational confirmation of this relation, postulated on the basis of sedimentation experiments by Rybenkov et al. [J. Mol. Biol. 267, 299 (1997)]. Such a relation was previously shown to hold with simulations for knot electrophoresis. We also show that there is an accurate linear dependence of s on the inverse of the radius of gyration R(g)(-1), more specifically with the inverse of the R(g) component that is perpendicular to the direction along which the polymer sediments. When the polymer sediments in a slab, the walls affect the results appreciably. However, R(g)(-1) remains to a good precision linearly dependent on n(c). Therefore, R(g)(-1) is a good measure of a knot's complexity.

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.

Scaling laws between the hydrodynamic parameters and molecular weight of linear poly(2-ethyl-2-oxazoline)

Poly(2-ethyl-2-oxazoline) (PEtOx), as an alternative polymer to poly(ethylene glycol), has potential applications in biomedical fields. The hydrodynamic parameters, such as the hydrodynamic radius and sedimentation coefficient, are important to understand its dynamics and properties, including its effect on the interactions between proteins and cells. In this study, we have investigated the hydrodynamic properties and thermodynamic parameters of a series of narrowly distributed PEtOx polymers with molecular weights ranging from 1.3 × 103 to 3.1 × 105 g mol−1, and the scaling laws between them by the use of a combination of analytical ultracentrifugation and laser light scattering. It is found that the sedimentation coefficient (s20,w) and hydrodynamic radius (Rh,0) at infinite dilution scale with molecular weight (Mw) as s20,w = Ks × Mαw = 0.0071 (S) × M0.462±0.019w and Rh,0 = KRh × Mβw = 0.0179 (nm) × M0.539±0.012w, respectively.

80 A new approach in determining the rigidity of nucleic acids and polymers

In polymer physics, persistence length of the chain molecules varying in degrees of stiffness is usually evaluated from hydrodynamic data for a set of polymers of different molecular mass М. It is known that from the extrapolation of translational diffusion coefficient to infinite molecular mass, one can calculate persistence length and hydrodynamic radius for rigid and semi-rigid molecules. If the persistence length of the molecule is much greater than its contour length, then the hydrodynamic parameters of the chain are independent of its size and such extrapolation is not applicable. In this case we proposed to plot the dependence M/s 0 2 (s 0 – sedimentation coefficient) or MD 2 (D – translational diffusion coefficient) vs. M (in the absence of volume effects) or n (number of monomers). In these coordinates the appearance of a plateau will correspond to the complete impermeability of the molecule and from plateau height the persistence length can be calculated. We analyzed literature data for a few synthetic polymers by our approach and determined their persistence lengths, the values of which were very close to those in the literature. Usually, the synthesis of polymer molecules with very high molecular mass is a very difficult task. But at the same time, natural polymers with high molecular mass are common in biology. A typical example is a DNA molecule. With increasing contour length of DNA, its conformation passes the next stages: (1) a rigid rod-like particle, (2) a semi-rigid permeable coil, and (3) an impermeable Gaussian coil (Serdyuk, Zaccai, & Zaccai, 2007). We analyzed by our approach sedimentation data of DNA molecules from the literature. It was shown that the plateau starts from 40,000 base pairs of the DNA molecule and the estimated value of persistence length was about 50 nm. This value is close to that from literature data (Lu, Weers, & Stellwagen, 2002). Thus, the proposed approach can be applied for flexibility analysis of biological macromolecules of different natures – from nucleic acids to proteins in strong denaturants.

Specific Charged Residues on the Myosin Heavy Chain (K368 and R406) Contribute to Head Interaction of Relaxed Smooth Muscle Myosin

Vertebrate smooth muscle contraction is regulated by phosphorylation of the myosin regulatory light chains. EM studies of smooth muscle myosin molecules suggest that in the dephosphorylated state, activity is switched off by asymmetric interactions between the two myosin heads, in which the “blocked” head is thought to be unable to bind to actin, while the “free” head has its ATPase activity inhibited. Comparative sequence analysis and molecular modeling suggest that the head interaction may be stabilized by charge attraction between D748 in the converter domain of the free head and K368 of the blocked head, and between R406 or K416 of the blocked head and E169 of the free head (Jung et al., MBC, 2008). To test these possibilities, we investigated the structure of expressed smooth muscle myosin and heavy meromyosin in which the charges at some of these sites was neutralized or reversed. While most of the mutants showed no obvious change in structure compared with wild type, HMM with charge reversal at both 368 and 406 showed fewer molecules with interacting heads when observed by metal shadowing. This was supported by a decrease in sedimentation coefficient (determined by analytical ultracentrifugation) and an increase in actin-activated ATPase activity of the mutant. Whole myosin with the same mutations formed filaments more readily than wild type, also consistent with these results. These observations suggest that charge interactions involving K368 and R406 play a role in stabilizing the head-head interaction of the inactive state. Interestingly, mutation of R403 in cardiac myosin (R406 in smooth muscle) increases motor activity of myosin and causes familial hypertrophic cardiomyopathy. Our results suggest that this could be in part a result of changes in head interaction.

Physical, Chemical, Biological and Biotechnological Sciences are Incomplete Without Each Other

By coupling of mechanics, optics, and mathematics, Theodor Svedberg invented the ultracentrifuge, which allowed separation of important biological materials by high centrifugal force, resulting in physical chemical separation and characterization of atherogenic low density lipoproteins and other biological molecules. Combining physics, chemistry and engineering, Ernest Lawrence invented the cyclotron, resulting in advancing nuclear physics, particle physics, molecular and materials science, and nuclear medicine. Using isotope– C-14, Melvin Calvin elucidated the photosynthesis cycle. Coupling photomultiplier tubes (PMTs) with NaI scintillation crystal, Hal Anger revolutionized nuclear imaging. Shashi Kumar and co-workers used biology and polymerase chain technology for the detection of mislabeled food materials. Combinations of biotechnology, chromatography, analytical chemistry and electron microscopy have resulted in organic chemists’ improved ability to synthesize isoprene compounds of interest. These are some of the examples that have moved science forward and demonstrate that physical, chemical, biological, and biotechnological sciences are incomplete without each other. The single most important advance in the use of centrifugal force to separate biologically important substances was the coupling of mechanics, optics and mathematics, by T. Svedberg and J.W. Williams in the 1920′s (http://humantouchofchemistry.com/theodorsvedberg.htm). The sedimentation coefficient is the rate at which particles of a given size and shape travel to the bottom of a tube under centrifugal force. The Svedberg unit is named after the Swedish chemist Theodor Svedberg (1884–1971), winner of the 1926 Nobel Prize in Chemistry for his work on colloids and his invention of the ultracentrifuge. Combining physics, chemistry and engineering Ernest Lawrence of the Lawrence Berkeley Laboratory invented Cyclotron (Fig 1). (http://www.nobelprize.org/nobel_prizes/physics/laureates/1939/lawrence-bio.html). The First Cyclotron, measuring only 5 inches in diameter, was constructed of glass, sealing wax and bronze; the material cost was about $25 (Fig 1). For his invention of the cyclotron, Lawrence was awarded the 1939 Nobel Prize in Physics.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Isolation and Characterization of Proteins from Chia Seeds (Salvia hispanica L.)

Chia ( Salvia hispanica L.) is a plant that produces seeds rich in some nutraceutical compounds with high protein content, but little is known about them; for this reason the aim of this study was to characterize the seed storage proteins. Protein fractions were extracted from chia seed flour. The main protein fraction corresponded to globulins (52%). Sedimentation coefficient studies showed that the globulin fraction contains mostly 11S and 7S proteins. The molecular sizes of all the reduced fractions were about 15-50 kDa. Electrophoretic experiments under native conditions exhibited four bands of globulins in the range of 104-628 kDa. The denaturation temperatures of crude albumins, globulins, prolamins, and glutelins were 103, 105, 85.6, and 91 °C, respectively; albumins and globulins had relatively good thermal stability. Selected globulin peptides by mass spectrometry showed homology to sesame proteins. A good balance of essential amino acids was found in the seed flour and globulins, especially of methionine+cysteine.

Titanium mineralization in ferritin: a room temperature nonphotochemical preparation and biophysical characterization

The incremental addition of titanium(III) citrate to H-chain homopolymers of human ferritin results in the formation of 1.5-6.5-nm particles of amorphous TiO(2) within the nanocage of the protein. The mineralization conditions are mild, featuring ambient temperature and no need for photochemical activation. Low ratios of titanium to protein favor intraprotein mineralization, and the products are characterized by stained and unstained transmission electron microscopy, UV-vis spectroscopy, dynamic light scattering, analytical ultracentrifugation, and metal analysis. With up to 1,000 equiv of metal, there is no change to the protein hydrodynamic radius or diffusion constant. There is, however, a systematic shift in the sedimentation coefficient, which confirms mineralization within the protein core.

NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys(3)-Cys(31), Cys(5)-Cys(20), and Cys(10)-Cys(30)) in the oxidized form (HD5(ox)). To probe the solution structure and oligomerization properties of HD5(ox), and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays. The NMR solution structure of HD5(ox), solved at pH 4.0 in 90:10 H(2)O/D(2)O, is presented (PDB: 2LXZ ). Relaxation T(1)/T(2) measurements and the rotational correlation time (τ(c)) estimated from a (15)N-TRACT experiment demonstrate that HD5(ox) is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19-21 (Val(19)-Cys(20)-Glu(21)) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7-14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5(ox) is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5(ox) at pH 7.0, supporting a tetrameric form ([HD5(ox)] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5(ox) dimer, were obtained. Millimolar concentrations of NaCl, CaCl(2), and MgCl(2) have a negligible effect on the HD5(ox) sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5(ox) in solution, and provide important insights into HD5(ox) structure/activity relationships and the requirements for antimicrobial action.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations.

Shaping and maintaining a medium-sized main channel in the Lower Yellow River

This paper studies relations between bankfull discharge, lateral cross section variation and the incoming flow and sediment condition in the Lower Yellow River using measured data from 1950 to 2003. Since 1950 the bankfull discharge has obviously decreased and the ratio of channel width to flow depth has increased. The critical annual average incoming sediment coefficient (defined as the ratio of sediment concentration to discharge) and discharge at the Huayuankou station are approximately 0.012 and 1,850 m3 s−1, respectively, for no accumulative deposition occurring in the reach from Huayuankou to Lijin. On this basis, a mathematical model is used to study the scale of the main channel in the Lower Yellow River and its corresponding bankfull discharge under possible incoming flow and sediment conditions in the near future. The main factors influencing the scale of the main channel are analyzed, and measures to shape and maintain a medium-sized channel are discussed. The results show the effect of various water and sediment combinations released from the Xiaolangdi Reservoir on the shaping of the main channel and suggest that under recent incoming flow and sediment conditions, it is possible to shape and maintain a medium-sized channel with a bankfull discharge of approximate 4,000 m3 s−1.

Multivalent di-nucleosome recognition enables the Rpd3S histone deacetylase complex to tolerate decreased H3K36 methylation levels

The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.

Dynamic Interaction of the Escherichia coli Cell Division ZipA and FtsZ Proteins Evidenced in Nanodiscs

The full-length ZipA protein from Escherichia coli, one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein, has been incorporated in single copy into nanodiscs formed by a membrane scaffold protein encircling an E. coli phospholipid mixture. This is an acellular system that reproduces the assembly of part of the cell division components. ZipA contained in nanodiscs (Nd-ZipA) retains the ability to interact with FtsZ oligomers and with FtsZ polymers. Interactions with FtsZ occur at similar strengths as those involved in the binding of the soluble form of ZipA, lacking the transmembrane region, suggesting that the transmembrane region of ZipA has little influence on the formation of the ZipA·FtsZ complex. Peptides containing partial sequences of the C terminus of FtsZ compete with FtsZ polymers for binding to Nd-ZipA. The affinity of Nd-ZipA for the FtsZ polymer formed with GTP or GMPCPP (a slowly hydrolyzable analog of GTP) is moderate (micromolar range) and of similar magnitude as for FtsZ-GDP oligomers. Polymerization does not stabilize the binding of FtsZ to ZipA. This supports the role of ZipA as a passive anchoring device for the proto-ring with little implication, if any, in the regulation of its assembly. Furthermore, it indicates that the tethering of FtsZ to the membrane shows sufficient plasticity to allow for its release from noncentral regions of the cytoplasmic membrane and its subsequent relocation to midcell when demanded by the assembly of a division ring.

Fibrinogen conformations and charge in electrolyte solutions derived from DLS and dynamic viscosity measurements

Hydrodynamic properties of fibrinogen molecules were theoretically calculated. Their shape was approximated by the bead model, considering the presence of flexible side chains of various length and orientation relative to the main body of the molecule. Using the bead model, and the precise many-multipole method of solving the Stokes equations, the mobility coefficients for the fibrinogen molecule were calculated for arbitrary orientations of the arms whose length was varied between 12 and 18nm. Orientation averaged hydrodynamic radii and intrinsic viscosities were also calculated by considering interactions between the side arms and the core of the fibrinogen molecule. Whereas the hydrodynamic radii changed little with the interaction magnitude, the intrinsic viscosity exhibited considerable variation from 30 to 60 for attractive and repulsive interactions, respectively. These theoretical results were used for the interpretation of experimental data derived from sedimentation and diffusion coefficient measurements as well as dynamic viscosity measurements. Optimum dimensions of the fibrinogen molecule derived in this way were the following: the contour length 84.7nm, the side arm length 18nm, and the total volume 470nm3, which gives 16% hydration (by volume). Our calculations enabled one to distinguish various conformational states of the fibrinogen molecule, especially the expanded conformation, prevailing for pH<4 and lower ionic strength, characterized by high intrinsic viscosity of 50 and the hydrodynamic radius of 10.6nm. On the other hand, for the physiological condition, that is, pH=7.4 and the ionic strength of 0.15M NaCl, the semi-collapsed conformation dominates. It is characterized by the average angle equal to <φ>=55°, intrinsic viscosity of 35, and the hydrodynamic radius of 10nm. Additionally, the interaction energy between the arms and the body of the molecule was predicted to be −4kT units, confirming that they are oppositely charged than the central nodule. Results obtained in our work confirm an essential role of the side chains responsible for a highly anisotropic charge distribution in the fibrinogen molecule. These finding can be exploited to explain anomalous adsorption of fibrinogen on various surfaces.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single-stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas-phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Stochastic simulation of structural properties of natively unfolded and denatured proteins

A new simulation strategy based on a stochastic process has been developed and tested to study the structural properties of the unfolded state of proteins at the atomistic level. The procedure combines a generation algorithm to produce representative uncorrelated atomistic microstructures and an original relaxation method to minimize repulsive non-bonded interactions. Using this methodology, a set of 14 unfolded proteins, including seven natively unfolded proteins as well as seven "classical" proteins experimentally described in denaturation conditions, has been investigated. Comparisons between the calculated and available experimental values of several properties, at hydrodynamic and atomic level, used to describe the unfolded state, such as the radius of gyration, the maximum length, the hydrodynamic radius, the diffusion coefficient, the sedimentation coefficient, and the NMR chemical shifts, reflect a very good agreement. Furthermore, our results indicate that the relationship between the radius of gyration and the hydrodynamic radius deviates from the Zimm's theory of polymer dynamics for random coils, as was recently observed using single-molecule fluorescent methods. Simulations reveal that the interactions between atoms separated by three chemical bonds (1-4 interactions) play a crucial role in the generation process, suggesting that the unfolded state is essentially governed by bonding and short-range non-bonding interactions.