Reassembled GFP: detecting protein-protein interactions and protein expression patterns.
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Animals↗
Biomedical subjects
Thomas J Magliery
Publications and source records attributed to Thomas J Magliery.
Sequence variation in ligand binding sites in proteins.
BACKGROUND: The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance. RESULTS: Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys2His2 zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies. CONCLUSION: Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites.
Binding Sites↗
Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism.
Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.
Amino Acid Sequence↗
Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif.
Consensus design methods have been used successfully to engineer proteins with a particular fold, and moreover to engineer thermostable exemplars of particular folds. Here, we consider how a statistical free energy approach can expand upon current methods of phylogenetic design. As an example, we have analyzed the tetratricopeptide repeat (TPR) motif, using multiple sequence alignment to identify the significance of each position in the TPR. The results provide information above and beyond that revealed by consensus design alone, especially at poorly conserved positions. A particularly striking finding is that certain residues, which TPR-peptide co-crystal structures show are in direct contact with the ligand, display a marked hypervariability. This suggests a novel means of identifying ligand-binding sites, and also implies that TPRs generally function as ligand-binding domains. Using perturbation analysis (or statistical coupling analysis), we examined site-site interactions within the TPR motif. Correlated occurrences of amino acid residues at poorly conserved positions explain how TPRs achieve their near-neutral surface charge distributions, and why a TPR designed from straight consensus has an unusually high net charge. Networks of interacting sites revealed that TPRs fall into two unrecognized families with distinct sets of interactions related to the identity of position 7 (Leu or Lys/Arg). Statistical free energy analysis provides a more complete description of "What makes a TPR a TPR?" than consensus alone, and it suggests general approaches to extend and improve the phylogenetic design of proteins.
Amino Acid Motifs↗
Detecting protein-protein interactions with GFP-fragment reassembly.
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Escherichia coli Proteins↗
A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics.
Combinatorial methodologies have revolutionized studies in biomolecular function, but they have so far proven less useful for understanding macromolecular structure and stability. This is largely because of the difficulty of screening libraries of molecules for biophysical properties, and the difficulty of interpreting structural effects in complicated molecules. Here, we report a novel, robust, cell-based screen for function of the four-helix bundle protein, Rop. By expression of green fluorescent protein from a ColE1 plasmid, the screen reports the copy number of the plasmid, which is modulated in Escherichia coli by Rop. We have engineered the screen so that the fluorescent phenotype can correspond to either Rop activity or lack thereof. We have used the screen to demonstrate with systematically constructed Rop core variants that not all molecules that bind small stem-loop RNAs in vitro are active in vivo. Rop is well understood from structural work and systematic mutations, which makes it possible to construct rational, targeted libraries. This screen makes it possible to rapidly interrogate such libraries effectively for proper protein folding and stability. In addition to its intended utility for combinatorial experiments in biophysics, the screen will allow further dissection of the mechanism of Rop-mediated plasmid copy number regulation in vivo.
Bacterial Proteins↗
Combinatorial approaches to protein stability and structure.
Why do proteins adopt the conformations that they do, and what determines their stabilities? While we have come to some understanding of the forces that underlie protein architecture, a precise, predictive, physicochemical explanation is still elusive. Two obstacles to addressing these questions are the unfathomable vastness of protein sequence space, and the difficulty in making direct physical measurements on large numbers of protein variants. Here, we review combinatorial methods that have been applied to problems in protein biophysics over the last 15 years. The effects of hydrophobic core composition, the most important determinant of structure and stability, are still poorly understood. Particular attention is given to core composition as addressed by library methods. Increasingly useful screens and selections, in combination with modern high-throughput approaches borrowed from genomics and proteomics efforts, are making the empirical, statistical correlation between sequence and structure a tractable problem for the coming years.
Catalysis↗
Library approaches to biophysical problems.
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Biophysical Phenomena↗
Exploring the limits of codon and anticodon size.
We previously employed a combinatorial approach to identify the most efficient suppressors of four-base codons in E. coli. We have now examined the suppression of two-, three-, four-, five-, and six-base codons with tRNAs containing 6-10 nt in their anticodon loops. We found that the E. coli translational machinery tolerates codons of 3-5 bases and that tRNAs with 6-10 nt anticodon loops can suppress these codons. However, N-length codons were found to prefer N + 4-length anticodon loops. Additionally, sequence preferences, including the requirement of Watson-Crick complementarity to the codon, were evident in the loops. These selections have yielded efficient suppressors of four-base and five-base codons for our ongoing efforts to expand the genetic code. They also highlight some of the parameters that underlie the fidelity of frame maintenance.
Anticodon↗
Expanding the natural repertoire of protein structure and function.
This review considers chemical and genetic approaches to the modification of protein structure. The historical interest in chemical and site-directed modifications will be briefly covered. Current chemical modification strategies will be presented. Biosynthetic mutagenesis with unnatural aminoacyl-tRNAs and current synthetic peptide ligation technologies will be covered in greater detail. The application of combinatorial genetic methods (e.g. phage display, DNA shuffling) to protein engineering with unnatural amino acids will be briefly discussed, with emphasis on the in vitro evolution of new enzymatic function (i.e. aminoacyl-tRNA synthetases). Throughout the review, the powerful insights gained from the combined use of these technologies will be illustrated by examples that focus on the elucidation of protein-ligand interactions.
Amino Acid Sequence↗