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P Thiyagarajan

Publications and source records attributed to P Thiyagarajan.

At least 19 recordsLinked to original sources
PubMed2004

Early collapse is not an obligate step in protein folding.

The dimensions and secondary structure content of two proteins which fold in a two-state manner are measured within milliseconds of denaturant dilution using synchrotron-based, stopped-flow small-angle X-ray scattering and far-UV circular dichroism spectroscopy. Even upon a jump to strongly native conditions, neither ubiquitin nor common-type acylphosphatase contract prior to the major folding event. Circular dichroism and fluorescence indicate that negligible amounts of secondary and tertiary structures form in the burst phase. Thus, for these two denatured states, collapse and secondary structure formation are not energetically downhill processes even under aqueous, low-denaturant conditions. In addition, water appears to be as good a solvent as that with high concentrations of denaturant, when considering the over-all dimensions of the denatured state. However, the removal of denaturant does subtly alter the distribution of backbone dihedral phi,psi angles, most likely resulting in a shift from the polyproline II region to the helical region of the Ramachandran map. We consider the thermodynamic origins of these behaviors along with implications for folding mechanisms and computer simulations thereof.

Acid Anhydride Hydrolases↗
PubMed2003

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme.

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

Animals↗
PubMed2003

Effect of hydrostatic pressure on closed-loop phase behavior of block copolymers.

The effect of hydrostatic pressure (P) on closed-loop phase behavior of deuterated polystyrene-block-poly(n-pentyl methacrylate) copolymers [dPS-PnPMA] was investigated by using small-angle neutron scattering and birefringence. For P<20.7 bar, dPS-PnPMA exhibited a lower disorder-to-order transition temperature (T(LDOT)) at 175 degrees C, and then an upper order-to-disorder transition temperature (T(UODT)) at 255 degrees C. With increasing pressure both T(LDOT) and T(UODT) were markedly changed, where dT(LDOT)/dP was 725 degrees C/kbar and dT(UODT)/dP was -725 degrees C/kbar. These are consistent with predictions by the Clausius-Clapeyron equation using measured values of the volume and enthalpy changes of both transitions. The large pressure coefficients imply that the closed-loop phase behavior observed for PS-PnPMA is an entropic-driven phase transition.

Journal Article↗
PubMed2002

Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.

Bacillus subtilis↗
PubMed2002

The rate-limiting step in the folding of a large ribozyme without kinetic traps.

A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions.

Base Sequence↗
PubMed2001

The thermodynamic origin of the stability of a thermophilic ribozyme.

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg(2+) to fold to their native structures that are very similar. The stability is measured as a function of Mg(2+) and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Base Sequence↗
PubMed2001

Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding.

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.

Kinetics↗
PubMed2001

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits.

Ribonuclease P (RNase P) catalyzes the 5' maturation of precursor tRNA transcripts and, in bacteria, is composed of a catalytic RNA and a protein. We investigated the oligomerization state and the shape of the RNA alone and the holoenzyme of Bacillus subtilis RNase P in the absence of substrate by synchrotron small-angle X-ray scattering and affinity retention. The B. subtilis RNase P RNA alone is a monomer; however, the scattering profile changes upon the addition of monovalent ions, possibly suggesting different interdomain angles. To our surprise, the X-ray scattering data combined with the affinity retention results indicate that the holoenzyme contains two RNase P RNA and two RNase P protein molecules. We propose a structural model of the holoenzyme with a symmetrical arrangement of the two RNA subunits, consistent with the X-ray scattering results. This (P RNA)2(P protein)2 complex likely binds substrate differently than the conventional (P RNA)1(P protein)1 complex; therefore, the function of the B. subtilis RNase P holoenzyme may be more diverse than previously thought. These revisions to our knowledge of the RNase P holoenzyme suggest a more versatile role for proteins in ribonucleoprotein complexes.

Avidin↗
PubMed2000

Mg2+-dependent compaction and folding of yeast tRNAPhe and the catalytic domain of the B. subtilis RNase P RNA determined by small-angle X-ray scattering.

We apply synchrotron-based small-angle X-ray scattering to investigate the relationship between compaction, metal binding, and structure formation of two RNAs at 37 degrees C: the 76 nucleotide yeast tRNA(Phe) and the 255 nucleotide catalytic domain of the Bacillus subtilis RNase P RNA. For both RNAs, this method provides direct evidence for the population of a distinct folding intermediate. The relative compaction between the intermediate and the native state does not correlate with the size of the RNA but does correlate well with the amount of surface burial as quantified previously by the urea-dependent m-value. The total compaction process can be described in two major stages. Starting from a completely unfolded state (4-8 M urea, no Mg(2+)), the major amount of compaction occurs upon the dilution of the denaturant and the addition of micromolar amounts of Mg(2+) to form the intermediate. The native state forms in a single transition from the intermediate state upon cooperative binding of three to four Mg(2+) ions. The characterization of this intermediate by small-angle X-ray scattering lends strong support for the cooperative Mg(2+)-binding model to describe the stability of a tertiary RNA.

Bacillus subtilis↗
PubMed1999

Solution structure of copper ion-induced molecular aggregates of tyrosine melanin.

Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D(2)O and H(2)O to study the structure of melanin aggregates formed in the presence of copper ions at various copper-to-melanin molar ratios. In the absence of copper ions, or at low copper ion concentrations, tyrosine melanin is present in solution as a sheet-like particle with a mean thickness of 12.5 A and a lateral extent of approximately 54 A. At a copper-to-melanin molar ratio of 0.6, melanin aggregates to form long, rod-like structures with a radius of 32 A. At a higher copper ion concentration, with a copper-to-melanin ratio of 1.0, these rod-like structures further aggregate, forming sheet-like structures with a mean thickness of 51 A. A change in the charge of the ionizable groups induced by the addition of copper ions is proposed to account for part of the aggregation. The data also support a model for the copper-induced aggregation of melanin driven by pi stacking assisted by peripheral Cu(2+) complexation. The relationship between our results and a previous hypothesis for reduced cellular damage from bound-to-melanin redox metal ions is also discussed.

Biophysical Phenomena↗
PubMed1996

Solution structures of GroEL and its complex with rhodanese from small-angle neutron scattering.

BACKGROUND: Molecular chaperonins 60 are cylindrical oligomeric complexes which bind to unfolded proteins and assist in their folding. Studies to identify the location of the protein substrate have produced contradictory results: some suggest that the substrate-binding site is buried within the interior of the complex, whereas others indicate an external (polar) location. RESULTS: Small-angle neutron scattering (SANS) measurements were made on GroEL chaperonin and on a complex of GroEL with rhodanese. The radius of gyration and the molecular weight determined from SANS measurements of GroEL agree well with those from its crystal structure. The positions of residues which were unresolved in the crystal structure have been confirmed. In addition, through model fitting of the SANS data, conformational changes in solution have been assessed and the location of bound rhodanese has been determined. CONCLUSIONS: The overall structure of GroEL in solution is similar to the crystal structure. In GroEL the N-terminal and C-terminal residues are organized compactly near the equator of the cylinder and the apical domains are flared by about 5 degrees. The best fit of SANS data suggests the existence of an equilibrium between the complex and single rings and monomers. SANS data for the GroEL-rhodanese complex are consistent with a model wherein one rhodanese molecule binds across the opening to the chaperonin cavity, rather than within it.

Binding Sites↗
PubMed1996

Probing self assembly in biological mixed colloids by SANS, deuteration, and molecular manipulation.

Small-angle neutron scattering was used to obtain information on the form and molecular arrangement of particles in mixed colloids of bile salts with phosphatidylcholine, and bile salts with monoolein. Both types of systems showed the same general characteristics. The particle form was highly dependent on total lipid concentration. At the highest concentrations the particles were globular mixed micelles with an overall size of 50A. As the concentration was reduced the mixed micelles elongated, becoming rodlike with diameter about 50A. The rods had a radial core-shell structure in which the phosphatidylcholine or monoolein fatty tails were arranged radially to form the core with the headgroups pointing outward to form the shell. The bile salts were at the interface between the shell and core with the hydrophilic parts facing outward as part of the shell. The lengths of the rods increased and became more polydispersed with dilution. At sufficiently low concentrations the mixed micelles transformed into single bilayer vesicles. These results give insight on the physiological function of bile and on the rules governing the self assembly of bile particles in the hepatic duct and the small intestine.

Bile Acids and Salts↗
PubMed1989

High-pressure small-angle neutron scattering (SANS) study of 1,2-dielaidoyl-sn-glycero-3-phosphocholine bilayers.

Small-angle neutron scattering of the trans-unsaturated DEPC has been investigated as a function of pressure at 12, 18.6 and 35 degrees C. A pressure-induced structural phase transition from a liquid-crystalline state to a gel state is observed at the temperatures studied. The critical pressure of this transition increases with increasing temperature with a delta P/delta T value of 51 bar/C degrees. The small-angle neutron scattering results indicate that the effect of the trans double bonds in DEPC is to enhance the conformational disorder in the hydrocarbon chains. In DEPC bilayers, a pressure-induced conformational ordering process is observed not only in the liquid-crystalline phase but also in the gel phase, which indicates that conformational disorder exists in the liquid-crystalline phase as well as in the gel phase.

Fatty Acids, Unsaturated↗
PubMed1987

Saturation-transfer electron paramagnetic resonance detection of anisotropic motion by sickle hemoglobin molecules in the polymer state.

The motional behavior of spin-labeled deoxygenated sickle hemoglobin has been studied by using both 9- and 35-GHz saturation-transfer electron paramagnetic resonance (EPR). Using spectral subtraction techniques and saturation-transfer EPR parameter correlation plots, we find that the saturation-transfer EPR spectra for the sickle hemoglobin gel state at high temperature and high hemoglobin concentration cannot be described as a simple superposition of spectra from immobilized hemoglobin plus solution-state hemoglobin but instead suggest that the individual sickle hemoglobin molecules exhibit limited, anisotropic, rotational oscillation within the polymer fiber. The spectra also imply that the symmetry axis for sickle hemoglobin rotational oscillation is approximately coincident with the nitroxide z axis of the covalently attached spin-label. We suggest that this anisotropic rotational motion may be produced by one or two of the known intermolecular contact sites within the sickle hemoglobin fiber acting as strong intermolecular binding sites, and producing "motional alignment" within the fiber; determining the location of the strong binding site should be important in focusing the future development of antisickling agents.

Electron Spin Resonance Spectroscopy↗