BARNASE BARSTAR SYSTEM PDF

Protein folding, protein design, hydrophobic core, barstar Abstract Barstar, the specific inhibitor of the bacterial ribonuclease barnase, is a small protein with a relatively large and compact hydrophobic core. To what extent does the arrangement of the secondary structure depend on the precise composition of this core? Selection from a large synthetic gene library, with the entire core replaced by a random selection of hydrophobic sidechains, has yielded a number of functional barstars. This suggests that designed novel secondary structure frameworks could be filled in similar fashion. Introduction Does the limited number of basic folds found in natural proteins imply that no other folds are possible, or as useful? Or is it simply that evolutionary success has not required the invention of other folds?

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Protein folding, protein design, hydrophobic core, barstar Abstract Barstar, the specific inhibitor of the bacterial ribonuclease barnase, is a small protein with a relatively large and compact hydrophobic core. To what extent does the arrangement of the secondary structure depend on the precise composition of this core?

Selection from a large synthetic gene library, with the entire core replaced by a random selection of hydrophobic sidechains, has yielded a number of functional barstars. This suggests that designed novel secondary structure frameworks could be filled in similar fashion. Introduction Does the limited number of basic folds found in natural proteins imply that no other folds are possible, or as useful?

Or is it simply that evolutionary success has not required the invention of other folds? A clear answer to this question could come from the design and construction of well-behaved proteins with folds unlike anything found in nature. How hard will this be? To start, for a well structured soluble protein, we want mostly hydrophilic sidechains on the outside and hydrophobics on the inside. This requirement implies that the distribution of hydrophobic residues along the amino acid chain must be one of the major determinants of the fold, as the precise fold determines which sidechains are pointed inwards from the backbone.

To design a new protein, one could build an external frame of secondary structural units, helices and sheets, each with one hydrophobic side, borrowed from known structures but arranged in a novel fold. With the interior hydrophobic residues randomized at the level of the synthetic gene, the interior three-dimensional jigsaw puzzle is then solved by screening a large library of peptide products for well-folded molecules.

Screening methods could include phage or ribosome display, by binding to monoclonal antibodies raised against elements of the exterior secondary structure as they appeared on the structures from which the were borrowed, or simply by selecting for protease resistance. Matsuura and Pluckthun [1] recently reported the start of a systematic program to answer the questions posed at the start of this paragraph, using similar procedures, starting with simple structures and building up to more complexity.

The results reported here support the idea that novel protein creation is feasible. According to Axe et al. Gassner et al [3] have reported that all ten residues of the major hydrophobic core of T4 lysozyme can be changed to methionine one at a time without ever losing all of its activity and the completely substituted molecule retained the basic fold of the wild type.

In a methods paper [4], I reported briefly on the isolation by phage display of genes for functional barstar with a compact eight-residue portion of its core so randomized.

Nor was I able to find any active barstars using the two-plasmid selection system described below. We have, then, three reports of successful refilling of hydrophobic cores of known structures. For the lysozyme experiments, methionine had been chosen because of its flexibility and its size. In a preliminary experiment, replacement of the entire residue core of barstar with methionines yielded no activity.

The choice of barstar [5] for this investigation was suggested by its small size, the relatively large size and compactness of its core, Fig. Figure 1: "Front" and "back" views of barstar. Sidechains of the hydrophobic core are represented as yellow space-filling spheres, the rest of the molecule as red ball and stick. Rasmol Methods and Results All of the methods outlined here have been described in detail elsewhere [4, 8]. Total synthesis of the barstar gene was carried out with the codons for all 22 of the hydrophobic core residues randomized to yield Leu, Val, Ile, Met or Phe.

This product was amplified by polymerase chain reaction PCR , using oligonucleotides Fig. The number of independent sequences originally produced was estimated by semi-quantitative PCR to be on the order of Also shown are the oligonucleotides used for PCR. The library was first ligated into the plasmid pMT, which carries chloramphenicol resistance, and transformed into E. The chloramphenicol resistant bacteria were tested by transformation by the compatible plasmid pMT [4,8].

This plasmid carries ampicillin resistance and an active barnase gene, making it lethal without concomitant synthesis of an active inhibitor. No successful transformants were found. This implies that functional barstars in the library are rare, estimated at less than about one in Genes for functional barstars were then selected by phage display. This phage displays multiple copies of the protein coded by the inserted gene on the phage surface. The product phage DNA was packaged into phage according to the supplier and amplified in E.

Selection of phage displaying active barnase inhibition was achieved by passing the spent E. Several cycles of this selection process were necessary to usefully concentrate phage carrying genuine barstar from a one in a million mix with wild type phage. The library was carried through eight such cycles. The prospective barstar genes from the selected phage were then transferred to the plasmid vector in E.

About half of these transformations were successful, and only the transformable clones produce barnase inhibitor measurable in extracts 9. The yields from these clones, however, were about two orders of magnitude less than obtained from the wild type barstar gene.

Activity of barnase decreased linearly to zero with addition of each of the active extracts, an indication of stable complex formation. Stability of three of the extracted inhibitors, numbers 13, 14 and 15 in Fig. Barnase inhibition in each of the four disappeared with a half life between 95 and minutes.

The inhibitor gene was sequenced in 33 of the plasmids which had tested positive. The identities of the 22 core residues of the twenty different sequences found are shown in Fig. Eleven sequences were found twice and three, three times, suggesting that the library contained no more positive sequences than a small multiple of the twenty found.

Discussion From Fig. Indeed, almost all positions can be occupied by a residue of any size, from the smallest, valine, to the largest, phenylalanine.

For the twenty sequences, the range of occurrence for each is 5 to 13 for leucine, 2 to 10 for valine, 0 to 5 for isoleucine, 0 to 5 for phenylalanine and 1 to 6 for methionine. The serine in number 10 is presumably from a PCR copy error.

The NTS codon Fig. Note also, however, the numbers in the right hand column of Fig. These represent the number of non-hydrogen C or S atoms in the sidechains of each core, approximately equivalent to the relative volumes they take up.

Unless the cores can maintain much more empty space than seems likely, this suggests that considerable distortion of the outside framework must be allowed. The three apparently much larger cores each have five phenylalanines and the flipping out of one of these would reduce the effective core volume. For barstar function, what must be maintained is only the configuration of the helix and adjacent loop that actually form the barnase binding site.

It seems likely, however, that this would require something fairly close to the natural fold of wild type barstar.

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