[86] Difference spectroscopy

Herskovits, Theodore T.

doi:10.1016/s0076-6879(67)11090-2

Cited by 192 publications

(87 citation statements)

References 67 publications

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“…The positive peak at 292 nm is characteristic for perturbation of tryptophans by a nondenaturing solvent additive like glycerol. Comparison of the magnitude of this change to that obtained for model tryptophan and tyrosine compounds indicated that both tryptophans are solvent-accessible (45).…”

Section: Physical Characterization Of the Procollagen Module Of Tsp1mentioning

confidence: 70%

“…Four scans were averaged for each measurement. The change in the extinction coefficient at 292 nm was used to estimate the exposure of the tryptophan residues as described by Herskovits (45). An equimolar mixture of the N-acetyl ethyl esters of tryptophan and tyrosine was used as the model compound.…”

Section: Expression and Purification Of The N-terminal Portion Of Thrmentioning

confidence: 99%

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Physical Characterization of the Procollagen Module of Human Thrombospondin 1 Expressed in Insect Cells

Misenheimer

Huwiler

Annis

et al. 2000

Journal of Biological Chemistry

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Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

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Section: Physical Characterization Of the Procollagen Module Of Tsp1mentioning

confidence: 70%

Section: Expression and Purification Of The N-terminal Portion Of Thrmentioning

confidence: 99%

Physical Characterization of the Procollagen Module of Human Thrombospondin 1 Expressed in Insect Cells

Misenheimer

Huwiler

Annis

et al. 2000

Journal of Biological Chemistry

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“…The difference in absorption between native TS and protein titrated with urea was recorded at 294 nm on a Hewlett Packard 8452A diode array spectrophotometer, using the tandem cell technique (Herskovitz, 1967). Equilibrium measurements were made in 20 mM KHP04, pH 7.0, 0.5 M KCl, 0.1 mM EDTA, 1 mM DTT (folding buffer), and varying urea concentration after all optical changes were complete.…”

Section: Methodsmentioning

confidence: 99%

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

et al. 1992

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Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.Keywords: dimerization; folding; oligomerization; thymidylate synthase Thymidylate synthase (TS) is a dimer of two identical, 35-kDa subunits (Kinemage 1) that catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,lO-methylenetetrahydrofolate (CH2H4folate) to dTMP and 7,8-dihydrofolate (H2folate). TS provides the only de novo pathway to deoxythymidine monophosphate (dTMP) production and has been studied extensively from the standpoints of structure, function, and inhibition (Santi & Danenberg, 1984). The crystal structure, which has been determined to 2.3 A resolution for Lactobacillus casei TS (Hardy et al., 1987;Perry et al., 1990; Finer-Moore, unpubl.), shows that the monomers form dimer contacts primarily between two, five-stranded beta sheets that maintain a unique +28" dihedral angle between strand directions (Kinemage 4). Three of these strands form abeta kink that creates part of the active site pocket of the second monomer. Further, two active site arginine residues (178' and 179') are donated from the opposing monomer and coordinate with the phosphate moiety of the substrate (Kinemage 3). Thus, the dimeric structure of TS is essential for the function of the enzyme.In order to study interactions at the dimer interface of TS, we sought conditions that would allow the separation, unfolding, and reassembly of subunits. To date, there is no evidence that the dimer can be dissociated and reformed. In the present work, we show that TS can be denatured to unfolded monomers in urea and subsequently refolded to its catalytically active native state. Results and discussionThe concentration of urea required to unfold TS was determined using UV and CD spectroscopies. The difference in environment of tryptophan residues between native and unfolded protein was monitored by the UV absorbance change at 294 nm (Fig. 1). Three zones were apparent along the equilibrium unfolding transition: (1) a range below 3.5 M urea where spectral changes were not observed, (2) a sharp transition between 3.5 M and 5.5 M denaturant, and (3) a range above 5.5 M urea where no further change in absorbance was detected. The AGHZ0 calculated for this equilibrium unfolding transition at 0 M urea was 19.1 kcal mol" (see Materials and methods). To ascertain whether secondary structure was lost at high urea concentration, the CD spectrum of TS equilibrated in 8.0 M urea was compared with that of native protein (Fig. 2). With...

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“…Ultraviolet Difference Spectra.-Matched tandem cells were used (Herskovits 1967) in a Perkin-Elmer 402 double-beam spectrophotometer. It is convenient to consider the results with respect to (i) the water-structuring ability of the ions used; (ii) the ability of ions to bind with the protein; and (iii) the ability of the ions to stabilize the native protein conformation.…”

Section: Methodsmentioning

confidence: 99%

Dehydration of Macromolecules II. Protective Effects of Certain Anions on Ribulosediphosphate Carboxylase Subjected to Low Water Potentials in Vitro

Steer¹

1973

Aust. Jnl. Of Bio. Sci.

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Studies of the instability of ribulosediphosphate carboxylase at low water potentials, generated in a pressure-membrane apparatus, have been continued. The presence of phosphate, pyrophosphate, citrate, or ribulose diphosphate during treatment protects the protein against fragmentation. Tris-sulphate, sodium sulphate, tetra-alkyl ammonium salts, or polyvinylpyrrolidone do not afford protection.The results are considered in terms of water-structuring by ions, the binding of ions to the protein, and stabilizing of the protein by ions. Reported effects of ions on enzyme kinetics do not explain the difference between sulphate and phosphate with respect to protection. It is concluded that protective ions must be bound to cationic sites on the protein but must also structure adjacent water. The additional structured water presumably prevents fragmentation of the protein that can occur at low water potentials.

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[86] Difference spectroscopy

Cited by 192 publications

References 67 publications

Physical Characterization of the Procollagen Module of Human Thrombospondin 1 Expressed in Insect Cells

Physical Characterization of the Procollagen Module of Human Thrombospondin 1 Expressed in Insect Cells

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

Dehydration of Macromolecules II. Protective Effects of Certain Anions on Ribulosediphosphate Carboxylase Subjected to Low Water Potentials in Vitro

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