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.

The low stability of natural proteins often limits their use in therapeutic, industrial and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for determining the approximate stabilities of protein variants at high throughput and low cost. The method is based on binding to a hydrophobic dye akin to ANS, which fluoresces upon binding to molten globules and thermal denaturation intermediates. No inherent properties of the protein, such as enzymatic activity or presence of an intrinsic fluorophore, are required. Very small sample sizes are analyzed using a realtime PCR machine, enabling the use of high-throughput purification. We show that the apparent T M values obtained from HTTS are approximately linearly related to those from CD thermal denaturation for a series of four-helix bundle hydrophobic core variants. We demonstrate similar results for a small set of TIM barrel variants. This inexpensive, general and scaleable approach enables the search for conservative, stable mutants of biotechnologically-important proteins, and it provides a method for statistical correlation of sequence-stability relationships.Natural proteins are often too unstable for therapeutic or industrial applications, or even for crystallography or directed evolution experiments. 1 There is still no reliable way to predict stabilizing mutations, and biophysical characterization of proteins is generally large-scale and low-throughput. 2 Except for enzymes, where enzymatic activity can be screened at elevated temperatures, high-throughput methods of screening for stability are lacking. Notably, the dominant classes of protein drugs-hormones, antibodies, cytokines, etc.-are binding proteins or ligands, not enzymes. Here we demonstrate that a dye-binding thermal shift screen, an extension of the ThermoFluor® method of screening for protein-ligand interactions, 3 reports the relative thermal stabilities of libraries of protein variants. We call the method HighThroughput Thermal Scanning, or HTTS.In ThermoFluor®, samples of a receptor protein are mixed with an analyte ligand and a fluorescent hydrophobic dye akin to 1-anilinonaphthalene-8-sulphonic acid (ANS). Folded proteins exclude these types of dyes, but molten globules and thermal denaturation intermediates bind them, resulting in a sharp increase in fluorescence. Binding of a ligand to the folded state of the receptor shifts the apparent melting temperature higher, which can be observed by heating the sample in a fluorimeter. Besides for drug discovery, this method has been applied to optimization of ligand and buffer conditions for crystallography. 4 We wished to invert the format of the screen, instead using a library of protein variants under the same conditions of dye and buffer, to probe the approximate relative thermal stabilities of the mutants. Since dye binding is so phy...

The detection of protein-protein interactions in vivo is of critical importance to our understanding of biological processes. The classic library approach has been to use the yeast two-hybrid screen, where an interaction between known bait and unknown prey proteins leads to restoration of transcription factor activity 1. However, its use is limited by host organism and nuclear localization requirements, and a tendency to detect indirect interactions (false positives). Bacterial two-hybrid screens have eliminated localization requirements and simplified many technical aspects of the procedure 2. An innovative approach has been the reassembly of protein fragments, which then directly report interactions. A suitable reporter protein is dissected at the genetic level, and the fragments are fused to bait and prey, which are then coexpressed in vivo. Bait and prey interaction brings the reporter fragments together, facilitating reassembly of the active reporter protein, giving a direct readout of the association. This method has been demonstrated for dihydrofolate reductase 3,4 , ubiquitin 5 and the green fluorescent protein6 (GFP) from Aequorea victoria. We recently described improvements to the original screen based on the reassembly of the GFP enhancedstability mutant sg100 in Escherichia coli 7. Our system, presented in the protocol that follows, consists of two plasmid vectors for the independent expression of fusions with N-and C-terminal fragments of GFP, and allows for simple visual detection of protein-protein interactions with a K D as weak as 1 mM. MATERIALS REAGENTS Plasmid vectors pET11a-link-NGFP and pMRBAD-link-CGFP and positive control vectors pET11a-Z-NGFP and pMRBAD-Z-CGFP are available upon request (Fig. 1) Primers for cloning into pET11a-link-NGFP and pMRBAD-link-CGFP (Fig. 1) Selective media: LB containing 100 µg/ml ampicillin and LB containing 35 µg/ml kanamycin Restriction enzymes: NcoI, BamHI, AatII, XhoI, XmaI and SphI (New England Biolabs (NEB)) Thermostable DNA polymerase (for example, Deep Vent Polymerase, NEB) dNTP solution (10 mM) Calf intestinal alkaline phosphatase (NEB) T4 DNA ligase (NEB) E. coli strains DH10B and BL21 (DE3), prepared as competent cells (preferably electrocompetent) Sequencing primers for pET11a-link-NGFP (T7 terminator primer 5′-tatgctagttattgctcag-3′) and pMRBAD-link-CGFP (5′ctactgtttctccatacccg-3′) ExoSAP-IT (USB) Screening medium: for 250 ml LB agar (for about ten plates) add 250 µl of 10 mM IPTG, 2.5 ml of 20% arabinose, 250 µl of 100 mg/ ml ampicillin (100 µg/ml) and 250 µl of 35 mg/ml kanamycin (35 µg/ml). Sterilize all additives by passing through a 0.2 µm filter. Lysis buffer: 50 mM Tris HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 1 mg/ml lysozyme, 0.5 mM CaCl 2 , 5 mM MgCl 2 Nucleases: DNase and RNase (Sigma) Ni-NTA agarose (Qiagen) Wash buffer: 50 mM Tris HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole EQUIPMENT Thermal cycler programmed with the desired amplification protocol Handheld long-wave UV lamp (365 nm) Disposable standing column (Bio-Ra...

General methods for selectively incorporating unnatural amino acids into proteins in vivo, directly from the growth media, would greatly expand our ability to manipulate protein structure and function. 1 For example, the ability to place fluorophores selectively into proteins in vivo would provide powerful tools for cell biology, or the ability to generate large quantities of proteins with metal binding or keto amino acids might lead to proteins with enhanced physical or catalytic properties. Our approach involves the generation of a suppressor tRNA/aminoacyl-tRNA synthetase (tRNA CUA /aaRS) pair that is orthogonal to Escherichia coli endogenous tRNA/synthetase pairs; that is, the orthogonal tRNA is not a substrate for any endogenous synthetases and the orthogonal synthetase does not recognize any endogenous tRNAs. 2,3 The specificity of this synthetase is then altered so that it charges the tRNA CUA only with a desired unnatural amino acid. One such orthogonal pair for use in E. coli was developed from the tRNA 2 Gln /GlnRS pair from Saccharomyces cereVisiae. 3 The development of additional orthogonal tRNA/aaRS pairs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Moreover, different aminoacyl synthetases may be better starting points for generating active sites with particular specificities (e.g., specificity for large hydrophobic vs small hydrophilic amino acids). To this end, we have analyzed biochemical data available for tRNA Tyr /TyrRS pairs from a variety of organisms. This analysis, together with in vivo complementation assays, has afforded a new orthogonal tRNA CUA Tyr /TyrRS pair as well as insights into the development of additional pairs.The identity elements of prokaryotic tRNA Tyr include a long variable arm in contrast to the short arm of eukaryotic tRNA Tyr . 4 In addition, eukaryotic tRNA Tyr contains a C1:G72 positive recognition element, whereas prokaryotic tRNA Tyr has no such consensus base pair. 5,6 In vitro studies have also shown that tRNA Tyr of S. cereVisiae 7 and Homo sapiens 8 cannot be aminoacylated by bacterial synthetases, nor do their TyrRS aminoacylate bacterial tRNA. To test whether tRNA CUA Tyr /TyrRS pairs from these organisms are orthogonal in E. coli, an in vivo complementation assay was used that is based on suppression of an amber stop codon in a nonessential position of the TEM-1 -lactmase gene encoded in plasmid pBLAM. 3 If the newly introduced suppressor tRNA CUA is aminoacylated by any endogenous E. coli synthetases, cells will grow in the presence of ampicillin. After expressing these tRNA CUA Tyr in E. coli strain DH10B transformed with pBLAM, cells survive at very high concentrations of ampicillin, greater than 1206 µg/mL (interpolated from IC 50 curves in Figure 1) for tRNA CUA Tyr derived from S. cereVisiae and 234 µg/mL for that from H. sapiens. When S. cereVisiae tRNA CUA Gln , which is an orthogonal tRNA, is tested under the same conditions, the cells survive at only 20 µg/mL ampicillin. 3 For comparison, E. coli str...

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an ''orthogonal'' suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA 2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA 2 Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotidedirected mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

Calculating protein stability and predicting stabilizing mutations remain exceedingly difficult tasks, largely due to the inadequacy of potential functions, the difficulty of modeling entropy and the unfolded state, and challenges of sampling, particularly of backbone conformations. Yet, computational design has produced some remarkably stable proteins in recent years, apparently owing to near ideality in structure and sequence features. With caveats, computational prediction of stability can be used to guide mutation, and mutations derived from consensus sequence analysis, especially improved by recent co-variation filters, are very likely to stabilize without sacrificing function. The combination of computational and statistical approaches with library approaches, including new technologies such as deep sequencing and high throughput stability measurements, point to a very exciting near term future for stability engineering, even with difficult computational issues remaining.

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