Use of a thermal inactivation technique to obtain binding constants for the Escherichia coli valyl-tRNA synthetase

Chuang, Hanson Y. K.; Bell, Fred E.

doi:10.1016/0003-9861(72)90245-7

Cited by 32 publications

(11 citation statements)

References 17 publications

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“…CLLD-DGK Has Enhanced Thermodynamic Stability-Improved kinetic stability can be partly a consequence of enhanced thermodynamic stability (13)(14)(15). We therefore compared the thermodynamic stability of CLLD-DGK with the wild-type protein using an assay we developed previously for DGK.…”

Section: Clld-dgk Has Enhanced Stability In Differentmentioning

confidence: 99%

Building a Thermostable Membrane Protein

Zhou¹,

Bowie²

2000

Journal of Biological Chemistry

142

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The poor stability of membrane proteins in detergent solution is one of the main technical barriers to their structural and functional characterization. Here we describe a solution to this problem for diacylglycerol kinase (DGK), an integral membrane protein from Escherichia coli. Twelve enhanced stability mutants of DGK were obtained using a simple screen. Four of the mutations were combined to create a quadruple mutant that had improved stability in a wide range of detergents. In n-octylglucoside, the wild-type DGK had a thermal inactivation half-life of 6 min at 55°C, while the quadruple mutant displayed a half-life of 35 min at 80°C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach should be applicable to other membrane proteins that can be conveniently assayed.Biochemical and structural studies of membrane proteins often require the proteins to be extracted from the lipid bilayer into a detergent micelle. Membrane proteins often exhibit unfavorable properties in detergent solution, however, such as poor solubility or stability (1, 2). Since the interactions between membrane proteins and detergents are very complex, the same protein can display different properties in different detergents. Thus, it is usually fruitful to screen for a detergent in which the protein of interest is active and stable. Unfortunately, many membrane proteins are only marginally stable and short-lived in even the best available detergent. In such cases, there are few alternatives for improving the experimental properties of the protein and the system can remain largely intractable.Recently, we discovered that single side-chain alterations can significantly improve the properties of the membrane protein diacylglycerol kinase (DGK) 1 from Escherichia coli, in detergent solution and that these stabilizing mutations can occur with high frequency. DGK is a 121-residue, trimeric enzyme, containing three transmembrane helices, that catalyzes the conversion of diacylglycerol and ATP to phosphatidic acid (3-7). We examined the stability of 20 different cysteine substitution mutants in DGK and found two mutations (I53C and I70C) that significantly enhanced the resistance of DGK to thermal inactivation. These results suggested that it might not be difficult to identify stabilizing mutations in a pool of random mutants (11). Here we show that enhanced stability mutants of DGK can be readily identified by screening a mutant library and that a robust membrane protein can be constructed by combining individual mutations. EXPERIMENTAL PROCEDURES Materials-The detergents n-octyl-␤-D-glucoside (OG), n-decyl-␤-Dmaltoside (DM), Cymal-5, and n-dodecyl-␤-D-maltoside(DDM)were obtained from Anatrace. LDAO and Empigen were obtained from Calbiochem. Cardiolipin and 1,2-sn-dioleoylglycerol (DAG) were obtained from Avanti Polar Lipids. All other chemicals were from Fisher or Sigma.Mutagenesis-We constructed a plasmid pSD005, containing a synthetic DGK gene. Plasmid pSD005 is identical to pSD004 (8), except that two mutatio...

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Section: Clld-dgk Has Enhanced Stability In Differentmentioning

confidence: 99%

Building a Thermostable Membrane Protein

Zhou¹,

Bowie²

2000

Journal of Biological Chemistry

142

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“…The rate constants of inactivation were determined in the absence (K o ) and presence (K ) of various SCN − concentrations. The protection constant (K p ) was then determined by the method of Chuang and Bell [32].…”

Section: Inactivation By Phmentioning

confidence: 99%

“…To determine the dissociation constant (K p ) of the enzyme-protector complex as described by Chuang and Bell [32], (K o kK ) when plotted against SCN − concentration yielded a hyperbolic plot as shown in Figure 4. The plot of 1\(K o kK ) against 1\[SCN − ] was linear and yielded a value of 24 µM for the dissociation constant of the enzyme-SCN − complex.…”

Section: Substrate Protectionmentioning

confidence: 99%

Mechanism-based inactivation of lacrimal-gland peroxidase by phenylhydrazine: a suicidal substrate to probe the active site

Mazumdar

Adak

Chatterjee

et al. 1997

Biochemical Journal

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Humans are exposed to various hydrazine derivatives for therapeutic control of several diseases, and mammalian peroxidases are implicated in the oxidative metabolism of many drugs. The results presented here indicate that lacrimal-gland peroxidase is irreversibly inactivated in a mechanism-based way by phenylhydrazine, which acts as a suicidal substrate in the presence of H # O # . The pseudo-first-order kinetic constants for inactivation at pH 5n5 are K i l 18 µM, k inact l 0n25 min −" and τ &! l 2n75 min, with a second-order rate constant of 0n75i10% M −" :min −" . Approx. 27 mol of phenylhydrazine and 54 mol of H # O # are required per mol of enzyme for complete inactivation. The pHdependent inactivation kinetics indicate the involvement of an ionizable group on the enzyme with a pK a value of 5n4, protonation of which favours inactivation. SCN − , the plausible physiological electron donor of the enzyme, protects it from inactivation. Binding studies by optical difference spectroscopy indicate that phenylhydrazine interacts with the enzyme with a K D value of 60 µM, and its binding is prevented by the presence of SCN − . The enzyme is also protected by 5,5-dimethyl-1-pyrroline N-oxide, a free-radical trap, suggesting the involvement of a radical species in the inactivation. ESR studies indicate the formation of a spin-trapped phenyl radical (a N l 15n9 G and

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“…If KO is defined as the apparent rate constant at zero concentration of protecting substrate and K , is defined as the apparent rate constant at concentration S of protecting substrate, a plot of (KO -K,) I against S p l gives a straight line with the intercept on the x axis given by -K p p l , where K is the equilibrium constant for the enzyme bin&ng the protecting agent (Chuang and Bell, 1972). In the case of the above protection by 6PGA the reciprocal plot for the determination of the I$, for 6PGA was indeed linear and gave a value of 18.2 p M for the equilibrium constant (Fig.…”

Section: Coenzyme and Substrate Bindingmentioning

confidence: 99%

Human Brain 6‐Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

Schofield

Edwards

1985

Journal of Neurochemistry

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6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.

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Use of a thermal inactivation technique to obtain binding constants for the Escherichia coli valyl-tRNA synthetase

Cited by 32 publications

References 17 publications

Building a Thermostable Membrane Protein

Building a Thermostable Membrane Protein

Mechanism-based inactivation of lacrimal-gland peroxidase by phenylhydrazine: a suicidal substrate to probe the active site

Human Brain 6‐Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

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