Biomedical subjects
Publications and source records attributed to G A Weiss.
Combinatorial libraries of alanine-substituted proteins can be used to rapidly identify residues important for protein function, stability and shape. Each alanine substitution examines the contribution of an individual amino acid sidechain to the functionality of the protein. The recently described method of shotgun scanning uses phage-displayed libraries of alanine-substituted proteins for high-throughput analysis.
A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.
Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.
We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.
Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.
We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.
Forty-three patients (47 heels) underwent decompression of the nerve to abductor digiti minimi with partial plantar fascia release for intractable plantar fasciitis over a 4-year period. Forty-one patients (45 heels) were available for follow-up. All of the patients had failed to respond to nonoperative treatment. The mean duration of symptoms before surgery was 34.8 months (range, 12-132 months), and the mean follow-up was 31.4 months (range, 11-66 months). Seventy percent of the patients in the study were overweight or obese. Before surgery, 39 patients (43 heels) rated their heel pain as severe. At follow-up, 34 of 45 (75.6%) of the heels were pain-free or only mildly painful. The mean visual analogue pain score dropped from 8.5 of 10 preoperatively to 2.5 of 10 postoperatively. Only four patients failed to report an improvement in their activity restrictions, and only one patient had a walking distance of under 100 m after surgery; this patient had been affected by a reflex sympathetic dystrophy. Overall, however, only 20 of 41 patients were totally satisfied with the outcome (48.8%). We recommend that the small group of patients who fail to respond to nonoperative treatment be considered for surgical intervention. The results in terms of symptomatic relief are generally good but in terms of patient satisfaction can only be rated as moderate. The patients should be counseled about the likely outcome of surgery.
The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.
BACKGROUND: Cytotoxic T-lymphocytes (CTLs) recognize complexes of short peptides with major histocompatibility complex (MHC) class I molecules. MHC molecules are polymorphic, and the products of different MHC alleles bind to different subsets of peptides. This is due to differences in the shape of the peptide-binding groove on the surface of the MHC protein, especially the 'pockets' into which anchor residues at each end of the peptide fit. Non-peptidic ligands for class I molecules may be useful clinically. RESULTS: By applying computer-aided design methods guided by X-ray structures, we designed and synthesized several MHC class I ligands, based on known peptide ligands, in which the tricyclic, aromatic compound phenanthridine replaced the central amino acids of the peptides. These semi-peptidic fluorescent ligands bound with high affinity and with allelic specificity to the peptide-binding groove of different MHC class I molecules, forming crystallizable complexes. CONCLUSIONS: Specificity for binding to different MHC class I molecules can be imparted to the common phenanthridine element by judicious choice of terminal peptidic elements from either nonamer or decamer peptides. The phenanthridine-based ligands have a long bound half-life, as do antigenic peptides.
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