Partially Folded Conformations in the Folding Pathway of Bovine Carbonic Anhydrase II: A Fluorescence Spectroscopic Analysis

Creatine kinase (CK, 1 EC 2.7.3.2) is a key enzyme for energy homeostasis in cells and plays a significant role in the transport of high energy phosphates via phosphocreatine to sites of ATP utilization in vivo (1-4). This enzyme catalyzes the reversible phosphoryl transfer between ATP and creatine (Cr) in the presence of Mg 2ϩ , and the release of an equimolar quantity of hydrogen ion (see Reaction I).Cr ϩ MgATP 2Ϫ º PCr 2Ϫ ϩ MgADP Ϫ ϩ H ϩ REACTION I

Section: Methodssupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid

Liang

Sanglier

et al. 2003

“…Phase Diagram Method of Fluorescence-The "phase diagram" method of fluorescence is a sensitive approach for the detection of unfolding/refolding intermediates of proteins (32)(33)(34)(35). The essence of this method is to build up the diagram of I( 1 ) versus I( 2 ), where I( 1 ) and I( 2 ) are the fluorescence intensity values measured on wavelengths 1 and 2 under different experimental conditions for a protein under going structural transformations.…”

Section: Determination Of Kinetic Constants For Lysozymementioning

confidence: 99%

Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Zhou

Liang

et al. 2004

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.The main question that protein folding encountered in vivo is that the intracellular environment is highly crowded because of the presence of high concentrations of soluble and insoluble macromolecules, which include proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides) (1-3). It has been estimated that the concentration of macromolecules in cytoplasm is in the range of 80 -400 g/liter (3-5), and all the macromolecules in physiological fluid media collectively occupy a lower limit of ϳ10% and a upper limit of ϳ40% of total fluid volume (1, 2, 6). These media are termed "crowded" or "volumeoccupied" rather than "concentrated" (7,8). The biophysical theory of macromolecular crowding has been well developed by a thermodynamic approach (2, 8 -10). The effect of excluded volume upon protein stability and conformation has been discussed using a simplified statistical thermodynamic model (11). One pertinent prediction of this model is that intramolecular excluded volume will increase the chemical potential of the unfolded state relative to that of the native or compact nonnative state, resulting in a relative stabilization of the native or compact non-native state relative to a fully unfolded state (12).Most of our knowledge about protein folding comes from biophysical studies of the refolding of pure denatured proteins at low concentrations in simple defined buffers carried out in the past three decades since the pioneering studies of Anfinsen (13). However, intra...

“…Phase Diagram Analysis of Fluorescence Intensity DataThe analysis of the unfolding data in terms of phase diagrams is known to be extremely sensitive to intermediate states (31)(32)(33)(34). Fig.…”

Section: Figmentioning

confidence: 99%

Cofactor Binding Modulates the Conformational Stabilities and Unfolding Patterns of NAD+-dependent DNA Ligases from Escherichia coli and Thermus scotoductus

Georlette

Blaise

Dohmen

et al. 2003

Self Cite

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD ؉ -dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD ؉ -DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD ؉ -dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.DNA ligases form a large family of evolutionarily related proteins that play important roles in a wide range of DNA transactions, including chromosomal DNA replication, DNA repair, and DNA recombination in all three kingdoms of life (1). Cofactor requirements divide the ligases into two subfamilies, the NAD ϩ -dependent DNA ligases and the ATP-dependent DNA ligases. Regardless of their energy source, they catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three distinct catalytic events (1). The first step involves activation of the ligase through the formation of a covalent adenylated intermediate by transfer of the adenyl group of NAD ϩ or ATP to the ⑀-NH 2 of a conserved lysine residue in the DNA ligase. In the second step the AMP moiety is transferred from the DNA ligase to the 5Ј-phosphate group at the single-strand break site, creating a new pyrophosphate bond. Finally, the phosphodiester bond formation is achieved upon an attack of the 3Ј-OH group of the DNA on the activated 5Ј-group with the concomitant release of AMP (1).At least one NAD ϩ -dependent DNA ligase (referred to as LigA) is found in every bacterial species (2). The bacterial LigA enzymes are of fairly uniform size (ϳ70 kDa) and display extensive amino acid sequence conservation throughout the entire protein (3-4). The atomic structures of the LigA enzymes from Bacillus stearothermophilus (N-terminal domain) (5) and Thermus filiformis (3) have been determined by x-ray crystallography. The catalytic core of the bacterial NAD ϩ -dependent DNA ligase con...