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Fast and slow conformational dynamics associated with enzyme catalytic cycles of Bacillus subtilis ribonuclease P protein in its folded state, bound to two sulfate anions are studied. The results reveal that the structural flexibility within the P protein ensemble might be significant for proper RNase P holoenzyme assembly and/or catalysis.

Ligation-state hydrogen exchange (HX) is used to study the intermediates in the P protein coupled folding and binding reaction and the ligand concentration dependence of the exchange rate is analyzed to discern regions of protection within each intermediate. The results indicate that the predominant route of HX is through the folded species and unliganded folded species, which represent only 0.45% and 0.0005% of the total protein population in 20mM sodium sulfate, respectively.

The background, physical basis and the experimental validation of a structural thermodynamic model of protein ensemble, known as COREX is reviewed. The COREX model reproduces a surprising number of apparently disparate biophysical and functional properties of proteins.

CD thermal denaturation studies of P protein in the presence of stabilizing solute of temperature-induced unfolding of the ligand-folded and the trimethylamine N-oxide (TMAO)-folded conformations of P protein. P protein is predominantly unfolded (D) at neutral pH and low ionic strength, however it folds upon the addition of sulfate anions as well as the osmolyte TMAO.

The authors review the publications on protein folding. The topics of interest include the methods for fast protein folding kinetics measuring and the analytical models used in interpreting these methods.

Research has been conducted on the yeast prolyl isomerase Ess 1. The folding of the Ess 1 domain and its binding to peptides that represent the C-terminal domain by circular dichroism and fluorescence has been investigated and discussed.

Research has been conducted on the ribonuclease P (RNase P). The biophysical properties of the Bacillus subtilis ribonuclease P protein subunit have been investigated to study the RNase P holoenzyme assembly.

A study was conducted to analyze the influence of a buried hydrogen bond to delta repressor folding kinetics. The conformational stability of each protein was determined using urea denaturation and by monitoring the state of the protein by circular dichroism. Results indicated that the folding of helix 3 is a critical slow step along the different folding pathways and occurs before the formation of the 14-77 hydrogen bond.

A monomeric lambda repressor containing a tryptophan at its core has been synthesized. The repressor is a truncated version of the N-terminal domain of the bacteriophage lambda cI protein. Its folding dynamics have been studied using fluorescence, stopped-flow and dynamic nuclear magnetic resonance. Results show that the folding occurs within the dead-time of the experiment in the absence of denaturants. The folding happened within 2 milliseconds in 3.4 M urea and 1.8 M GdmCl. Stopped-flow and dynamic NMR results agree on the two-state nature of the folding with a folding time in submilliseconds at 20 degrees C.

A study was conducted to examine the dynamics of fast-folding proteins such as a monomeric lambda-repressor to describe the order and rate of events associated to their folding mechanism. The diffusion-collision model was utilized in describing the protein folding dynamics and in estimating the intrinsic alpha-helix propensities. Results revealed the folding mechanism was distributed among various pathways which were highly sensitive to amino acid sequence.

The structures of the heat and cold denatured states of monomeric bacteriophage lambda repressor are thermodynamically and conformationally the same. The aromatic 1H nuclear magnetic resonance (NMR) spectra of the urea denatured and thermally denatured states are the same at 37 degrees Celsius. The circular dichroism and NMR studies of thermal and urea denaturation of residues 6-85 of the N-terminal region of lambda repressor indicate that the totally denatured state is the sole non-native state at temperatures over 25 degrees Celsius.

The cleavage of double-stranded DNA oligomers by d-sigma* to d-sigma excited state of Pt2(P2O5H2(super 2-))(sub 4)(super 4) (1), in the presence of light generates an even ladder of phosphate terminated bands. The cleavage is effective in the presence of Mg2+ ions and O2. The products obtained are consistent with the abstraction of 4' and 5' hydrogens from deoxyribose motifs as revealed by electrophoresis gel analysis. A high resolution footprint having discerned binding between gamma repressor and DNA helix is obtained by photoreaction of 1.

The basic steps involved in protein folding can occur on a submillisecond scale, as evidenced by nuclear magnetic resonance studies of monomeric gamma repressor. This urea-dependant molecule is more exposed to urea than previously studied transition states, and its quick folding time may be made possible by its all-helical secondary structure.

Alpha-helices in proteins often undergo further folding to form coiled coils, which play an important role in conformational changes in proteins. Coiled coil transitions occur in proteins such as hemagglutinin, heat shock factors and other DNA-binding proteins like the dimeric eukaryotic transcription factors. Many coiled coil structures contain discontinuities whose biological roles are not yet clear. New powerful computational tools and recent developments in experimental molecular biology will prove useful for the further elucidation of coiled coils and their discontinuities.

The nuclear magnetic resonance spectroscopy technique is employed to identify the folding intermediate associated with the Bacillus subtilis RNase P protein. The results demonstrate the presence of an equilibrium, as well as kinetic intermediate in the folding mechanism of the protein.

The bacterial RNase P holoenzyme from Bacillus subtilis (P protein) is used as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The utility of trimethylamine N-oxide (TMAO), an osmolyte that has stabilized the unliganded folded form of the protein, has enabled the analysis of the folding process of P protein in the absence of ligand.

Thermodynamic and kinetic models are developed to understand the folding mechanism of multiple independently-folding domain (MIFD) proteins by examining the folding behavior of fused double B domain sequences of staphylococcal protein A (SpA). It is observed that the global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein, and that the global unfolding rate of a MIFD protein might be many orders of magnitude slower than that of the constituent domains.

A comparison between the folding kinetics, stability and occurrence preferences of N-cap and helix C-cap is conducted for examining their role in protein folding and for reconciliation of the differences in C-cap preferences between helical peptides and proteins. Increased stability, accelerated folding and slower unfolding were observed after the introduction of consensus, in-register N-capping motifs.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A is carried out to identify the folding and unfolding dynamics of this small three-helix bundle protein. Investigation of data obtained provides significant understanding of the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.