Supplementary Materials: Amberlite XAD-4 is a convenient tool for removing Triton X-100 and Sarkosyl from protein solutions.pdf

Supplementary Materials</p

Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from <i>Mycobacterium tuberculosis</i>

Mycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis–Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site

Overexpression and purification of HoLaMa W866F.

(A) SDS-PAGE of protein extracts isolated from E. coli subjected to induction, at 30°C, of HoLaMa W866F or W924F. M, NI, I tot, and I sol indicate: i) molecular mass markers; ii) total proteins extracted from not induced culture; iii) total proteins extracted from induced culture; iv) soluble proteins extracted from induced culture. The molecular masses of the markers are indicated at the left. (B) SDS-PAGE of protein extracts isolated from E. coli subjected to induction, at 15°C, of HoLaMa W866F. M, NI, I tot, and I sol indicate: i) molecular mass markers; ii) total proteins extracted from not induced culture; iii) total proteins extracted from induced culture; iv) soluble proteins extracted from induced culture. The molecular masses of the markers are indicated at the left. (C) SDS-PAGE of protein extracts isolated from E. coli subjected to induction, at 15°C, of HoLaMa W924F. M, NI, I tot, and I sol indicate: i) molecular mass markers; ii) total proteins extracted from not induced culture; iii) total proteins extracted from induced culture; iv) soluble proteins extracted from induced culture. The molecular masses of the markers are indicated at the left. (D) SDS-PAGE of fractions eluted from a HiTrap Blue column (5 mL) and containing purified HoLaMa W866F. M, I, and FT indicate molecular mass markers, input, and flow-through, respectively. Fraction numbers and the molecular masses of the markers are indicated at the top and at the left, respectively.</p

Folding to unfolding transition of HoLaMa as induced by urea.

Intrinsic fluorescence of HoLaMa was determined exciting samples at 280 nm and detecting emission at 328 nm. (A) Folded to unfolded transition of 500 nM HoLaMa as induced by the addition of urea. (B) Fluorescence of HoLaMa tryptophanes as affected by binding of the enzyme to the 40mer polyA DNA (see Fig 3). The enzyme concentration was 500 nM. (C) Folded to unfolded transition of HoLaMa bound to the 40mer polyA DNA (see Fig 3) as induced by the addition of urea. Enzyme and DNA concentration were 500 nM each. All the samples were in 50 mM sodium phosphate buffer (pH 8.0), 50 mM NaCl. The continuous lines represent the best fits of the equation described by Pace and Shaw [31] to the experimental data.</p

DNA binding by HoLaMa W866F.

Stopped-flow assay of HoLaMa W924 fluorescence changes triggered by 3.2 μM 40mer polyA DNA binding, in the presence of 3.4 μM W866F HoLaMa. The continuous line represents the best fit to the experimental observations of a single exponential equation. Residuals are the differences between the observed values and those expected according to the kinetic model used to fit the data.</p

Cartoon of the DNA substrates used in the present work.

Cartoon of the dsDNAs used in elongation assays performed in the presence of HoLaMa, Klenow, or Klenow exo-. The sequences of the template strands are also reported.</p

High-resolution one-dimensional proton NMR spectroscopy of HoLaMa.

Panel (A): spectrum acquired at 25°C using 50 μM HoLaMa dissolved in 50 mM sodium phosphate buffer containing 50 mM NaCl at pH 8.0. (B) Detail of the aliphatic part of the spectrum. (C) Detail of the aromatic and NH-backbone signal region.</p

Kinetics of DNA extension catalysed by HoLaMa, Klenow, or Klenow exo^- enzyme.

(A) Elongation of the 1 μM 30mer polyA DNA in the presence of 100 μM dTTP. The reactions were catalysed by 360 nM HoLaMa (green line), 30 nM Klenow (magenta line) or 30 nM Klenow exo- (blue line). (B) Activity of 360 nM HoLaMa at the expense of 1 μM 30mer 7AC DNA, in the presence of: 100 or 200 μM dTTP (green and blue lines, respectively), dTTP and dGTP 100 μM each (magenta line), dTTP and dATP 100 μM each (cyan line), or 200 μM dGTP (red line). (C) Extension of 30mer 7AC (green line) and 30mer 3AC (blue line) DNA by 360 nM HoLaMa, in the presence of 100 μM dTTP. (D) Kinetics of reactions catalysed by 360 nM HoLaMa (green line), 30 nM Klenow or Klenow exo- (magenta and blue lines, respectively) at the expense of 1 μM 30mer 7AC DNA, in the presence of dTTP and dGTP 100 μM each. The reaction catalysed by HoLaMa is the same shown in (B) and is reported here for comparison. (E, F) Electrophoresis analysis of DNA elongation reactions catalysed by HoLaMa. Electrophoresis of aliquots of reaction mixtures (20 μL) containing 1.5 μM 30mer 7AC DNA (see Fig 3), 360 nM HoLaMa, 100 μM dTTP and 100 μM dGTP (E) or 200 μM dTTP only (F). Both electrophoretic runs were performed using TBE-urea gels (15% polyacrylamide) subjected to constant voltage (200 V) for 40 min.</p

Sensitivity of HoLaMa and Klenow enzymes to the competitive inhibition exerted by dGTP.

(A) Assay mixtures containing 360 nM HoLaMa (empty circles) or 30 nM Klenow exo- (filled circels) in 100 mM Tris (pH 8.0), 5 mM MgCl2, 0.25 mM inosine, were used to test the extension of 1 μM 30mer polyA DNA. Reactions were started by the addition of 100 μM dTTP, and the pyrophosphate released by the polymerases was determined using a previously described enzyme-coupled assay [28]. The competitive inhibition, if any, triggered by dGTP on the action of both HoLaMa and Klenow exo- was evaluated at concentrations ranging from 20 to 100 μM of dGTP. (B) Activity of 360 nM HoLaMa at the expense of 10 μM 17mer 1AC DNA (see Fig 3), in the presence of: 100 μM dTTP (green line), or dTTP and dGTP 100 μM each (magenta line). (C) Activity of 30 nM Klenow exo- at the expense of 10 μM 17mer 1AC DNA, in the presence of: 100 μM dTTP (green line), or dTTP and dGTP 100 μM each (magenta line).</p

A Synthetic Post-transcriptional Controller To Explore the Modular Design of Gene Circuits

The assembly from modular parts is an efficient approach for creating new devices in Synthetic Biology. In the “bottom-up” designing strategy, modular parts are characterized in advance, and then mathematical modeling is used to predict the outcome of the final device. A prerequisite for bottom-up design is that the biological parts behave in a modular way when assembled together. We designed a new synthetic device for post-transcriptional regulation of gene expression and tested if the outcome of the device could be described from the features of its components. Modular parts showed unpredictable behavior when assembled in different complex circuits. This prevented a modular description of the device that was possible only under specific conditions. Our findings shed doubts into the feasibility of a pure bottom-up approach in synthetic biology, highlighting the urgency for new strategies for the rational design of synthetic devices