Calculation of protein extinction coefficients from amino acid sequence data

Gill, Stanley C.; Hippel, Peter H. von

doi:10.1016/0003-2697(89)90602-7

Cited by 5,347 publications

(3,815 citation statements)

References 10 publications

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“…Tus concentrations were determined from its UV absorption spectrum using 280 ) 39 700 M -1 cm -1 (29). Protein samples for ESI-MS were prepared by diluting the protein stock to 0.5 mg/mL in water, followed by extensive dialysis against 0.1% formic acid.…”

Section: Methodsmentioning

confidence: 99%

Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

et al. 2000

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The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K D ) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K D measured for the binding of wild type and His 6 -Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K D by 200-300-fold, whereas the A173T substitution increased K D by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

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Section: Methodsmentioning

confidence: 99%

Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

et al. 2000

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“…Protein concentration was determined spectrophotometricaUy using the molar extinction coefficients at 280 nm (15930 M -1 cm -1 for TF and 6970 M -1 cm -1 for TF fragment) which were calculated from the primary structure according to Gill and Von Hippel [23].…”

Section: Protein Analysismentioning

confidence: 99%

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Stoller¹,

Tradler²,

Rücknagel³

et al. 1996

FEBS Letters

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The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cisltrans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatieally active fragment could be isolated after proteolysis by subtifisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni 2+-NTA column. Subsequent digestion with subtifisin and anionexchange chromatography yielded a TF fragment encompassing amino acids Gin-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 ~1-1 s -l could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrofide FK506.

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“…The concentrations of the chimeras were calculated from the absorbances at 280 nm in 6 M guanidine-hydrochloride as described (Gill & von Hippel, 1989). The 2 methods of protein estimation yielded values within +5% of each other.…”

Section: Protein Estimationmentioning

confidence: 99%

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

et al. 1994

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 a-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability , Protein Sci 2:1472-1481). To further probe the structurefunction relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of a-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

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Calculation of protein extinction coefficients from amino acid sequence data

Cited by 5,347 publications

References 10 publications

Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

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