Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA~~b,,/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to "75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for I9F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.

Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mib-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mib-CK were significantly inhibited under the same conditions. At low chloride concentrations, the Vmax of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 11-fold increase in Km(PCr), but only 1.7- and 2.8-fold reductions in Vmax, respectively, compared to wild-type Mib-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (i.e., Kd > Km). We confirmed this finding for wild-type Mib-CK by determining the Kd and Km values for both substrates in the forward reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20^25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.z 2000 Federation of European Biochemical Societies.

The denaturant-induced equilibrium unfolding of octameric mitochondrial creatine kinase, dimeric cytosolic muscle-type creatine kinase, and monomeric arginine kinase was investigated. Stable unfolding intermediates for all three enzymes were manifested by a strongly biphasic red shift of intrinsic protein fluorescence upon increasing denaturant concentrations. In the intermediate state, all proteins were monomeric and enzymatically inactive, but still retained a globular shape. Native tertiary structure interactions were largely disrupted, while at least 50% of the secondary structures were conserved, as suggested by near- and far-UV circular dichroism, respectively. A significantly increased surface hydrophobicity of the intermediate conformation, compared to both the native and the fully unfolded states, was observed by the binding of the hydrophobic fluorescent dye ANS. The observed properties agree formally with the definition of the molten globule state, but can be alternatively explained by a sequential unfolding of individual domains, involving a transient exposure of domain interfaces. Very similar unfolding profiles for all three proteins suggest that the formation of stable unfolding intermediates is not a consequence of the specific oligomeric structures of the CKs but rather due to a common, probably two-domain architecture of the guanidino kinase protomers.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mi,-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in 296% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC; Mib-CK .creatine.MgADP .NO,-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK.Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimerdimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.