Biomedical subjects

F H Arnold

Publications and source records attributed to F H Arnold.

79 records · Page 5Linked to original sources

PubMed1990

Engineering enzymes for non-aqueous solvents.

Enzymes may be redesigned to permit catalysis in non-aqueous solvents by engineering their amino acid sequences, thereby altering their physical and chemical properties to suit the new solvent environment. The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in non-aqueous media. These approaches are illustrated by several examples where protein engineering has resulted in enzymes that are better suited for catalysis in organic solvents.

Amino Acid Sequence↗

PubMed1989

Metal affinity precipitation of proteins.

Proteins containing multiple surface-accessible histidine residues can be precipitated using small quantities of bis-copper chelates. The chelates serve to crosslink the proteins, presumably via the accessible histidines, leading to the formation of large, insoluble complexes. When excess copper chelate is used to carry out the precipitation, the resulting precipitate has a stoichiometry of 1:1 copper:accessible histidine. The precipitation is analogous to antibody-antigen precipitin reactions and can be described qualitatively using simple equilibrium theory developed for those systems. Human hemoglobin contains a large number of surface histidines and is efficiently precipitated by the copper salt CuSO4 as well as by bis-copper chelates. Sperm whale myoglobin contains many fewer surface histidines and is precipitated only by the bis-chelates. The effects of the number of accessible histidines on the protein, the chain length separating the two chelates, and the pH on the precipitation reaction have been investigated.

Animals↗

PubMed1988

Protein design for non-aqueous solvents.

Improving protein stability in unnatural and suboptimal environments is a promising application of protein engineering technology. Carefully designed amino acid alterations may lead to dramatic positive effects on the stability of proteins under highly perturbing conditions, such as in non-aqueous solvents. Applications of biocatalysts and proteins with specific binding capabilities in the chemical industry have been severely limited by constraints placed on the solvent environment. With the advent of convenient methods for altering the amino acid composition and even synthesizing entirely new protein molecules, it is worthwhile to consider engineering proteins for stability in non-aqueous solvents. In order to identify the features that a protein would need for stability in organic media, we have been studying the structure and properties of the hydrophobic protein crambin. Crambin is unique in that it is soluble and stable in very high concentrations of polar organic solvents. Crambin and its water-soluble homologs offer a powerful demonstration of protein engineering for non-aqueous solvents. This paper describes the structural features that contribute to crambin's special properties. Based on these observations and consideration of how non-aqueous solvents affect the interactions important in protein folding, a set of rules for designing non-aqueous solvent-stable proteins is proposed.

Amino Acid Sequence↗

PubMed1987

Structure, dynamics, and thermodynamics of mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2.

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.

Base Sequence↗

PubMed1986

Analytical affinity chromatography. II. Rate theory and the measurement of biological binding kinetics.

Affinity chromatography can be used to measure equilibrium constants and kinetics of biological interactions. The local-equilibrium theory presented in the preceding paper is extended to include mass transfer and kinetic effects. Solutions for both zonal and frontal elution are presented. For highly nonlinear isotherms, the frontal elution method is preferred. Experiments with bovine serum albumin binding to immobilized Reactive Blue show that the binding kinetics inside the porous gel are several orders of magnitude slower than typical biological binding reactions in solution. The temperature dependence of the kinetic constants indicate that the binding may still be diffusion-controlled.

Animals↗

PubMedDate not supplied

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.

Chemical Phenomena↗

PubMedDate not supplied

Functional expression of horseradish peroxidase in E. coli by directed evolution.

In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET-22b(+) vector (Novagen) as a fusion to the signal peptide PelB. A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild-type HRP gene in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate)). However, weak peroxidase activity was detected in the supernatant in the absence of IPTG. The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96-well microplate format. From 12 000 clones screened in the first generation, one was found that showed 14-fold higher HRP activity than wild-type, amounting to approximately 110 microg of HRP/L, which is similar to that reported from laborious in vitro refolding. No further improvement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H2O2. These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli. This approach will greatly facilitate efforts to "fine-tune" those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking.

Biotechnology↗