Biomedical subjects

J S Fetrow

Publications and source records attributed to J S Fetrow.

9 recordsLinked to original sources

PubMed1996

Loop replacement and random mutagenesis of omega-loop D, residues 70-84, in iso-1-cytochrome c.

To study the role of omega loop D, residues 70-84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.

Amino Acid Sequence↗

PubMed1995

A method of directed random mutagenesis of the yeast chromosome shows that the iso-1-cytochrome c heme ligand His18 is essential.

A method to perform site-directed random mutagenesis directly in the yeast chromosomal DNA at the iso-1-cytochrome c-encoding gene locus (CYC1) is described. To test the effectiveness of the random mutagenesis procedure, the heme ligand His18 was mutated to Ala (H18A), rendering cytochrome c (Cyc) nonfunctional. Random mutagenesis was performed by transforming yeast cells with a synthetic oligodeoxyribonucleotide (oligo) that randomizes the codon for His18. The transformed cells were then selected for reversion to a functional Cyc on selective media. Ten functional mutants were recovered, all of which had integrated the synthetic oligo. Sequencing showed that five of the recovered mutants carried the His codon, CAU, and five mutants contained the His codon, CAC. Because Arg had previously been found as a heme ligand, this mutant was produced by standard techniques and integrated into the yeast chromosome. These yeast did not produce a holo cytochrome c that was detectable by low-temperature spectroscopy. To develop a selection for nonfunctional Cyc, competent yeast (which lack the ability to synthesize tryptophan) were cotransformed with a plasmid carrying the TRP1 gene and the random oligo, and were plated on media lacking tryptophan. Of the 1200 colonies that grew, 120 tested negative for the integration of the random oligo, demonstrating that this particular selection for nonfunctional protein is not feasible. A method is thus described for directed, random mutagenesis directly in the yeast chromosome that can be used to probe structure/function relationships in Cyc. Only His can act as a heme ligand at position 18, using the functional selection described here.

Base Sequence↗

PubMed1995

A proposed structural model of domain 1 of fasciclin III neural cell adhesion protein based on an inverse folding algorithm.

Fasciclin III is an integral membrane protein expressed on a subset of axons in the developing Drosophila nervous system. It consists of an intracellular domain, a transmembrane region, and an extracellular region composed of three domains, each predicted to form an immunoglobulin-like fold. The most N-terminal of these domains is expected to be important in mediating cell-cell recognition events during nervous system development. To learn more about the structure/function relationships in this cellular recognition molecule, a model structure of this domain was built. A sequence-to-structure alignment algorithm was used to align the protein sequence of the fasciclin III first domain to the immunoglobulin McPC603 structure. Based on this alignment, a model of the domain was built using standard homology modeling techniques. Side-chain conformations were automatically modeled using a rotamer search algorithm and the model was minimized to relax atomic overlaps. The resulting model is compact and has chemical characteristics consistent with related globular protein structures. This model is a de novo test of the sequence-to-structure alignment algorithm and is currently being used as the basis for mutagenesis experiments to discern the parts of the fasciclin III protein that are necessary for homophilic molecular recognition in the developing Drosophila nervous system.

Algorithms↗

PubMed1995

Omega loops: nonregular secondary structures significant in protein function and stability.

Omega (omega-) loops, a nonregular secondary structure found in globular proteins, are characterized by a polypeptide chain that follows a loop-shaped course in three-dimensional space. They do not contain repeating backbone dihedral angles or regular patterns of hydrogen bonding; however, many omega-loops contain a large number of hydrogen bonds, therefore it is not correct to think of omega-loops as structures lacking in hydrogen bonds. omega-Loops are found almost exclusively at the protein surface and exhibit amino acid preferences consistent with this observation. Since the first description of omega-loops in 1986, experiments have been conducted to probe the role of these structures in protein function, stability, and folding. It has become clear that omega-loops are often involved in protein function and molecular recognition. One motif, an omega-loop lid, that is flexible and mobile until substrate or inhibitor is bound and which probably plays a role in one or more steps of enzymatic catalysis, has been described in a variety of enzymes. Because they lack the periodic hydrogen bonding patterns of the regular secondary structures, some omega-loops are well suited for such functional roles in proteins. However, loops with a higher-than-average number of hydrogen bonds or hydrophobic contacts may play roles in protein stability or folding. Rather than determining further geometric definitions of loops, it may be instructional to group them according to their roles in protein structure, i.e., as categories of functional omega-loops, stability omega-loops, and folding omega-loops.

Amino Acids↗

PubMed1994

A molecular model of the inducer binding domain of the galactose repressor of Escherichia coli.

The C-terminal inducer binding domain of the Escherichia coli galactose repressor (GalR) is homologous to several periplasmic chemoreceptor proteins whose three-dimensional structures have been determined at high resolution (Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5226-5237; Mowbray, S. L. and Cole, L. B. (1992) J. Mol. Biol. 225, 155-175). The protein backbone was constructed from the coordinates of glucose/galactose-binding protein using the Homology program (Biosym Technologies, San Diego). Loops were built by searching for substructures in the structure data base, and the side chains were built using a rotamer-base program. A small amount of energy minimization relieved steric strain within the model. The GalR model that has been constructed is consistent with the principles of protein structure; values obtained for the compactness and buried surface area of the model compare favorably with those determined for the chemoreceptor protein structures. The model is consistent with, and provides structural interpretations for, experimental results obtained from physical and biochemical studies. Predictions are made concerning the residues conferring the specificity of galactose induction of GalR and for self-association to dimers. The model provides a first step toward correlating the structure and regulation of GalR and in determining the chemistry of its homologous and heterologous interactions that are critical to its role as a regulator of transcription initiation.

Amino Acid Sequence↗

PubMed1993

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.

Amino Acid Sequence↗

PubMed1993

New programs for protein tertiary structure prediction.

Prediction of protein tertiary structure remains an unsolved problem in molecular biology, but a solution to this problem is extremely important for protein engineering and rational drug design. Recent developments in motif recognition and side chain modeling present the prospect of nearly automatic model building for a large fraction of newly determined protein sequences. We review some of these new algorithms and present preliminary results of their application to the prediction of a structure for fasciclin III, a neural adhesion molecule from Drosophila.

Animals↗

PubMed1993

Automatic derivation of substructures yields novel structural building blocks in globular proteins.

Because the general problem of predicting the tertiary structure of a globular protein from its sequence is so difficult, researchers have tried to predict regular substructures, known as secondary structures, of proteins. Knowledge of the position of these structures in the sequence can significantly constrain the possible conformations of the protein. Traditional protein secondary structures are alpha-helices, beta-sheets, and coil. Secondary structure prediction programs have been developed, based upon several different algorithms. Such systems, despite their varied natures, are noted for their universal limit on prediction accuracy of about 65%. A possible cause for this limit is that traditional secondary structure classes are only a coarse characterization of local structure in proteins. This work presents the results of an alternative approach where local structure classes in proteins are derived using neural network and clustering techniques. These give a set of local structure categories, which we call Structural Building Blocks (SBBs), based upon the data itself, rather than a priori categories imposed upon the data. Analysis of SBBs shows that these categories are general classifications, and that they account for recognized helical and strand regions, as well as novel categories such as N- and C-caps of helices and strands.

Cluster Analysis↗

PubMed1989

Deletions and replacements of omega loops in yeast iso-1-cytochrome c.

omega (omega)-loops are protein secondary structural elements having small distances between segment termini. It should be possible to delete or replace certain of these omega-loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae were investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different omega-loops were individually deleted, or in which one omega-loop was replaced with five different segments. Deletions encompassing amino acid positions 27-33 and 79-83 either prevented synthesis of the holoprotein, or produced highly labile iso-1-cytochromes c, whereas deletions encompassing positions 42-45 and 48-55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue positions 24-33) with same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso-1-cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.

Amino Acid Sequence↗