The Unfolding Thermodynamics of c-Type Lysozymes: A Calorimetric Study of the Heat Denaturation of Equine Lysozyme

Griko, Yuri V.; Freire, Ernesto; Privalov, George; Dael, H. Van; Privalov, Peter L.

doi:10.1006/jmbi.1995.0510

Cited by 103 publications

(103 citation statements)

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“…3A and Table 1). C p for the native state was linear with temperature (dC p /dT ϳ 0.27 kcal K Ϫ1 mol Ϫ1 ) as expected (29,30). As shown in Fig.…”

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confidence: 61%

“…3A, at high temperatures the C p of MsPimA approaches the value of the completely unfolded state calculated from the known sequence, assuming that all amino acid residues are exposed to water (29). Because the partial heat capacity of a protein is a very sensitive indicator of the exposure of protein groups to water (29,30), it can be concluded that upon completion of the large heat absorption peak, MsPimA is completely unfolded. Thus, MsPimA unfolds with a small total heat capacity incre-…”

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confidence: 99%

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Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria

Guerin¹,

Schaeffer

Chaffotte

et al. 2009

Journal of Biological Chemistry

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Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the ␤-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic ␣-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response, host-pathogen interactions, and tumor cell metastasis. Most of the enzymes encoded in eukaryotic/prokaryotic/archaeans genomes that are responsible for the biosynthesis and modification of glycan structures are GTs 3(1). Here we have focused in the phosphatidyl-myo-inositol mannosyltransferase A (PimA), an essential enzyme of mycobacterial growth that initiates the biosynthetic pathway of key structural elements and virulence factors of Mycobacterium tuberculosis, the phosphatidyl-myo-inositol mannosides (PIM) lipomannan and lipoarabinomannan (2-5). This amphitropic enzyme catalyzes the transfer of a Manp residue from GDPMan to the 2-position of PI to form phosphatidyl...

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“…3A and Table 1). C p for the native state was linear with temperature (dC p /dT ϳ 0.27 kcal K Ϫ1 mol Ϫ1 ) as expected (29,30). As shown in Fig.…”

mentioning

confidence: 61%

mentioning

confidence: 99%

Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria

Guerin¹,

Schaeffer

Chaffotte

et al. 2009

Journal of Biological Chemistry

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“…Standard activity assays of these samples showed that ;90% of the original activity was retained. During thermal denaturation of HEWL, the hydrophobic core of the protein is exposed, but the disulfide bonds remain intact Khechinashvili et al, 1973;Privalov & Khechinashvili, 1974;Griko et al, 1995;Ibara-Molero & Sanchez-Ruiz, 1997!. Intermolecular association of the hydrophobic core of proteins leads to aggregation and precipitation.…”

Section: Resultsmentioning

confidence: 99%

Protein renaturation by the liquid organic salt ethylammonium nitrate

2000

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The room-temperature liquid salt, ethylammonium nitrate~EAN!, has been used to enhance the recovery of denaturedreduced hen egg white lysozyme~HEWL!. Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg0mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.

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“…On the other hand, the remaining activity of lysozyme with 1. increased with incubation time, and reached the equilibrium at 2 and 7 min, respectively. In thermal denaturation of lysozyme without protein aggregation, when the hydrophobic core of proteins is exposed, but the disulfide bonds keep intact, denatured proteins spontaneously refold to their native structures on cooling after thermal denaturation [26][27][28][29][30]. The refolding of thermally-denatured proteins is enhanced in the presence of protic ionic liquids such as alkylammonium nitrate and alkylammonium formates [24,25].…”

Section: Thermal Inactivation Of Lysozymementioning

confidence: 99%