Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.

The existence of parallel pathways in the folding of proteins seems intuitive, yet remains controversial. We explore the folding kinetics of the homodimeric Escherichia coli toxin CcdB (Controller of Cell Division or Death B protein) using multiple optical probes and approaches. Kinetic studies performed as a function of protein and denaturant concentrations demonstrate that the folding of CcdB is a four-state process. The two intermediates populated during folding are present on parallel pathways. Both form by rapid association of the monomers in a diffusion limited manner and appear to be largely unstructured, as they are silent to the optical probes employed in the current study. The existence of parallel pathways is supported by the insensitivity of the amplitudes of the refolding kinetic phases to the different probes used in the study. More importantly, interrupted refolding studies and ligand binding studies clearly demonstrate that the native state forms in a biexponential manner, implying the presence of at least two pathways. Our studies indicate that the CcdA antitoxin binds only to the folded CcdB dimer and not to any earlier folding intermediates. Thus, despite being part of the same operon, the antitoxin does not appear to modulate the folding pathway of the toxin encoded by the downstream cistron. This study highlights the utility of ligand binding in distinguishing between sequential and parallel pathways in protein folding studies, while also providing insights into molecular interactions during folding in Type II toxin-antitoxin systems.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api’s activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

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Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.conditional mutants | rational design | cold sensitivity | heat-induced expression | transfer between organisms C onditional mutants are powerful tools for studying gene function in vivo. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and lacks that function under a different set of conditions, called restrictive, whereas the wild type (WT) phenotype is similar across both conditions. Cold-sensitive (cs) mutants behave like loss-of-function mutants at temperatures lower than a cutoff temperature, but have WT-like phenotypes at higher temperatures. In contrast, temperature-sensitive (ts) mutants show heat sensitivity and behave like the WT below the restrictive temperature. Both cs and ts mutants can provide important information about protein structure, function, and assembly. cs mutants are rarer than ts mutants, and the molecular basis for generation of cs phenotypes is currently unclear.cs mutants have been used to analyze various biological phenomena, most commonly the cell cycle (1-3), ribosome assembly (4-8), and protein export (9-11), as well as to understand macromolecular structure-function relationships (12, 13). Various ts and cs variants also have helped us understand P22 phage coat protein assembly. Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20). Cold sensitivity also has been attribut...

pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional 15 N-1 H heteronuclear singlequantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.

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A 19-amino acid long proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) is encoded in the fruit fly genome. Native Dro is glycosylated at a specific threonine residue, but the non-glycosylated peptide retains antibacterial activity. Dro shows sequence similarity to several other PrAMPs that bind in the ribosomal nascent peptide exit tunnel and inhibit protein synthesis by varying mechanisms. However, the target and mechanism of action of Dro remain unknown. Here we show that the primary mode of Dro action is inhibition of termination of protein synthesis. Our in vitro and in vivo experiments demonstrate that Dro stalls ribosomes at stop codons, likely sequestering class 1 release factors associated with the terminating ribosome. As the result, Dro, at subinhibitory concentrations, strongly promotes readthrough of stop codons. The elucidated mode of Dro action allows assigning it as the second member of the type II PrAMPs, of which only one representative, the antimicrobial peptide apidaecin (Api) produced by honeybees, was previously known. However, despite its functional similarity with Api, Dro interacts with the target in a markedly distinct way. The analysis of a comprehensive single-amino acid substitution library of endogenously expressed Dro variants shows that binding to the ribosome involves interactions of multiple amino acid residues distributed through the entire length of the PrAMP. Our data further show that the ribosome-targeting activity of non-glycosylated Dro can be significantly enhanced by single amino acid substitutions illuminating directions for improving its antibacterial properties.