Expression and characterization of ¹³FNIII and VWFA2 tandem repeat proteins.

<p>Superposed elution profiles from size exclusion chromatography of (¹³FNIII)_2–8 proteins (A) and (VWFA2)_6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)_n unfolding curves in the presence and absence of Ca²⁺.</p

Assembly of chimeric constructs and one-pot concatamer formation.

<p>(A) Assembly of (FNIII)₂-VWFA2-(FNIII)₂ sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037617#pone.0037617.s002" target="_blank">Table S1</a></b>) show that the A2 synthon is sandwiched between two ¹³FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.</p

Efficient synthon assembly with split-and-pool reactions.

<p>(A) Equimolar amounts of BsaI or BsmBI deprotected ¹³FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (¹³FNIII)₁ to (¹³FNIII)₈ repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (¹³FNIII)₈ construct.</p

Split-and-pool assembly of DNA synthons.

<p>(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.</p

Apparent melting temperature of concatameric proteins.

<p>Apparent melting temperature of concatameric proteins.</p

Vectors for synthon recombination and transfer to expression plasmids.

<p>(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (<b>1</b>) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (<b>2</b>) to yield a composite product (<b>3a</b>), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (<b>3b</b>). Final assemblies (<b>3a</b> and <b>4</b>) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.</p

Characterization of the Mycobacterial Acyl-CoA Carboxylase Holo Complexes Reveals Their Functional Expansion into Amino Acid Catabolism

<div><p>Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these - the AccD1-AccA1 complex from <i>Mycobacterium tuberculosis</i> with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α−β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.</p></div

Growth phenotype characterization and complementation of mycobacterial accD1 and accA1 genes.

<p>(A) Growth of M. smegmatis wt and knockout strains in minimal 7H9 media supplemented with isovalerate (IVAL). The wt is in green; ΔaccD1-ΔaccA1 is in red; ΔaccD2-ΔaccA2 (control) is in blue. Error bars are in black. (B) Rescue of the M. smegmatis growth phenotype observed in isovalerate-containing minimal media by complementing ΔaccD1-ΔaccA1 (blue line) with the M. tuberculosis homolog of AccD1-AccA1 (red line). The wt is in green. Average values for the culture density were calculated from four replicates.</p

Identification and biochemical characterization of the AccD1-AccA1 substrate 3-methylcrotonyl-CoA.

<p>(A) Untargeted CoA-profiles of YCC knockout strains. Mass spectra of Co-A profiles of M. smegmatis wt and knockout strains. Insets show spectra at dashed line. The peak evident in the ΔaccD1-ΔaccA1 spectrum is that of 3-methylcrotonyl-CoA. (B) Steady-state kinetics of AccD1-AccA1 incubated with 3-methylcrotonyl-CoA, propionyl-CoA and acetyl-CoA. (C) Time course of the consumption of 3-methylcrotonyl-CoA by purified M. tuberculosis AccD1-AccA1 and AccD5-AccA3.</p

Mycobacterial YCC protein/protein interactions.

<p>(<i>A</i>) Binary, hetero-dimeric protein/protein interactions are indicated by double circles when found reciprocally, by using reverse bait/prey protein pairs, and by simple circles when identified only in one of the two possible combinations. Homo-oligomeric assemblies were found for all proteins tested (dotted patterns). Mycobacterial YCC CT subunits AccD1 to AccD6 are denoted “D1” to “D6” (grey), BT subunits AccA1 to AccA3 are denoted “A1” to “A3” (no background), and the AccE ε-subunit is labeled “E” (black). (<i>B</i>) Identification of YCC holo complexes. Arrows point to the protein component used as prey; protein/protein assemblies, for which binary interactions were identified by the reverse use of bait/prey are indicated by thick double arrows. For further details, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004623#ppat.1004623.s002" target="_blank">S2 Table</a>.</p