Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid

Liang, Yi; Du, Fei; Sanglier, Sarah; Zhou, Bing-Rui; Xia, Yi; Dorsselaer, Alain Van; Maechling, Clarisse; Kilhoffer, Marie‐Claude; Haiech, Jacques

doi:10.1074/jbc.m304050200

Cited by 59 publications

(36 citation statements)

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“…Theoretically, with a K D of 50 nM and a total protein concentration of 10 M, 93% should be in the form of dimer. When studied by nanoESI-MS under native-like conditions, almost full dimerization (89%) was observed (Figure 1a), which has also been found by other groups [19]. The interaction strength of the two monomer units is thus large enough to survive the ionization and transmission processes.…”

Section: Resultssupporting

confidence: 52%

“…When studying noncovalent complexes by ESI-MS, it is known that by increasing the ionic strength or adding organic solvents to the aqueous buffer will affect the hydrophobic effect in solution [17]. Moreover, the intermolecular interaction forces studied by ESI-MS are sensitive to other effects, such as changes in temperature [18], solution pH [19], and source pressure [20], which can also lead to dissociation of noncovalent complexes. …”

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confidence: 99%

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Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Bich

Baer

Jecklin

et al. 2010

J. Am. Soc. Mass Spectrom.

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The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real "snap-shot" of the situation in solution can be obtained. (J Am Soc Mass Spectrom 2010, 21, 286 -289) © 2010 American Society for Mass Spectrometry E lectrospray is an exceptionally soft ionization technique, which has allowed the observation of numerous noncovalent complexes, including protein-protein, protein-small molecule, and protein-DNA complexes [1,2]. A major question is still whether the information obtained from gas-phase results is representative of the species present in solution. The term "noncovalent bonding" in biochemistry generally summarizes three types of intermolecular forces: electrostatic interactions (e.g., salt bridges), dipolar interactions (e.g., hydrogen bonds), and van der Waals interactions (e.g., hydrophobic interactions) [3]. In addition, protein-water interactions often play a major role in complex stabilization. In solution, hydrophobic interactions are very weak compared with the hydrogen bonds between water molecules. Hence, it is not the hydrophobic forces that are the actual reason for the interaction of nonpolar binding partners, but rather the surrounding water molecules that "force" hydrophobic binding partners to aggregate. This solvent-driven process is called the hydrophobic effect, and plays a major role in protein folding, adhesion of lipid bilayers, partitioning effects of drugs and metabolites, and many other aggregation phenomena in chemistry and biology [4][5][6]. The hydrophobic effect is defined by the thermodynamics of mixing hydrophobic compounds with water, and is dominated by large, entropic factors [4,7]. Obviously, in the absence of a hydr...

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Section: Resultssupporting

confidence: 52%

mentioning

confidence: 99%

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Bich

Baer

Jecklin

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…In principle, 1 and 2 are arbitrary wavelengths of the fluorescence spectrum, but in practice such diagrams will be more informative if 1 and 2 are on different slopes of the spectrum, such as 320 and 365 nm (32)(33)(34)(35). It has been noted that application of this method to protein unfolding/refolding predicts that the dependence of I 320 versus I 365 will be linear if changes in the protein environment lead to an all-or-none transition between two different conformations.…”

Section: Determination Of Kinetic Constants For Lysozymementioning

confidence: 99%

“…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

Journal of Biological Chemistry

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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...

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“…Thermodynamics of the Binding of Zn 2ϩ to Tau-ITC provides a direct route to the complete thermodynamic characterization of non-covalent, equilibrium interactions (32,43), and DTT concentrations as low as 1 mM can cause severe baseline artifacts due to background oxidation during the titration. Therefore ITC was used to measure the binding affinity of Zn 2ϩ to Tau monomer in the absence of DTT.…”

Section: Cys-291 and Cys-322 Are Key Residues In The Interaction Of Znmentioning

confidence: 99%