Purification of ribonuclease T1

Cn, Pace; Gr, Grimsley; Bj, Barnett

doi:10.1016/0003-2697(87)90186-2

Cited by 22 publications

(15 citation statements)

References 19 publications

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“…We used this ratio to monitor our success in removing a yellow pigment during the purification of RNase TI (Pace et al, 1987). At the time, we thought it might be possible to use the t values in water to model the exposed chromophores in a protein, and the t values in propanol to model the buried chromophores in a protein and that this might allow us to calculate E values for proteins with reasonable accuracy.…”

Section: Predicting the Absorption Coefficient Of A Proteinmentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .

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Section: Predicting the Absorption Coefficient Of A Proteinmentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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“…Early efforts attempted to increased stability by decreasing the entropy of the DSE. These approaches typically involved the insertion of disulfides or the use of Gly to Ala and X to Pro substitution [38][39][40]. Problems arose if strain was introduced into the native state or if the modification inadvertently stabilized a compact denatured state.…”

Section: Denatured State Interactions As a Target For Protein Designmentioning

confidence: 99%

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Cho¹,

Sato²,

Horng³

et al. 2008

Archives of Biochemistry and Biophysics

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

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“…Disulfide bridges are also considered to contribute to stability by decreasing the entropy of the unfolded state [202,203]. The production of active enzymes having correctly formed disulfide bonds, introduced by site-directed mutagenesis, has been reported.…”

Section: Disulfide Bridgesmentioning

confidence: 99%

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

Vriend¹,

Eijsink²

1993

J Computer-Aided Mol Des

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Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new techniques have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

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Purification of ribonuclease T1

Cited by 22 publications

References 19 publications

How to measure and predict the molar absorption coefficient of a protein

How to measure and predict the molar absorption coefficient of a protein

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

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