Conformational analysis by near-UV circular dichroism.

<p>Near-UV CD spectra were recorded at 25°C with 0.5 mg/ml of protein in 20 mM sodium phosphate pH 8. While all mutants had a strong minimum at 292 nm, M4c (red curve) had a weak signal throughout the range studied, suggesting loss of packing near aromatic residues.</p

Impact of the new interloop salt bridge on EstGtA2 thermostability under alkaline pH.

<p>A. Structural alignment of the interloop portion for Est30 (blue) and LipS (pink). B. Structural alignment for MGL-H257 (green) and EstGtA2 (white), salt bridge at the conserved interloop position is shown for respective structures. Arrow shows change reversal for MGL-H257 and EstGtA2 (N’ enzymes). The proteins (wild type or H222R mutant) were incubated for 10 min at the indicated temperature and the residual activity on <i>p</i>NP-octanoate (compared with non-incubated enzymes) was measured at 25°C in 20 mM CAPS buffer pH 10.</p

Activity profile for wild type and mutants.

<p>Specific activity (µmol min^-1 mg^-1) was measured from 25 to 65°C for the wild type and mutants in 20 mM sodium phosphate pH 8 using <i>p</i>NP-octanoate as substrate. The initial rates (V₀) were measured below substrate saturation and than reflect <i>K</i>_cat/<i>K</i>_m conditions. Each point (specific activity values) was obtained from three different experiments and reported relative to wild type at 50°C (optimal conditions). Standard deviations do not exceed 5%. The wild type is shown as close circles and bold line. The curve for the quadruple mutant M4a is shown in orange and in red for M4c. The mutation R37A enhances the activity and shift the optimal temperature compared to wild type, the relative activity at 25 versus 60°C are listed in Table 1.</p

Activity and stability dependence on pH for EstGtA2.

<p>The apparent melting temperatures (derived from CD) as function of pH are shown as closed circles. Open circles show relative activity (hydrolysis of <i>p</i>NP-octanoate) at different pH values at 50°C (optimal temperature). Standard deviations for <i>T</i>_<i>m</i> ranged between 0.1-0.3°C and do not exceeded 5% for activity. Buffers used were sodium citrate/citric acid (pH4-5), sodium phosphate (pH 6-8), Tris-HCl (pH 9) and CAPS/NaOH (pH 10-11).</p

Thermal unfolding of EstGtA2 and mutants.

<p>Thermal unfolding curves recorded from 25 to 90°C were expressed as fraction folded derived from the CD signal at 222 nm as function of temperature. Samples were made of 0.5 mg/ml of protein in 20 mM sodium phosphate pH 8. EstGtA2 had the highest denaturation temperature among all versions studied. The quadruple mutant M4b is the least stable. The quadruple mutant M4a is shown in orange.</p

Distinctive salt bridge patterns conserved within family XV and XIII.

<p>Panel A: Multiple sequence alignment for N’ subfamily enzymes (MGL from <i>Bacillus</i> sp. H-257, <i>G</i>. <i>thermoleovorans</i>, <i>G</i>. <i>kaustophilus</i> and EstGtA2 from <i>G</i>. <i>thermodenitrificans</i>) and representative of the LipS (4FBL) and N subfamily (1TQH). The secondary structure elements from MGL H-257 (3RM3) are shown on top. The cap domain is boxed (thick line). Residue numbering is based on EstGtA2 and MGL H-257. The residues of the catalytic triad are identified by a triangle above the MSA. The seven N’ conserved salt bridges: E3-R54, E12-R37, E66-R140, H110-E78, D124-K178, H197-D148 and D205-R220 are identified by close triangles below the MSA with same letter. Residues shaved by alanine-scanning are identified by a black box below the MSA. Open triangles refer to LipS salt bridges pattern. The tolerant interloop salt bridge located in (<i>i</i> -2, <i>i</i> -4) from the catalytic Asp and His respectively is identified with a red star above the MSA. The alanine or arginine substitutions are indicated by a box A or R. Panel B: Multiple sequences alignment of N-related subfamily enzymes, compared to LipS and MGL (from N’ subfamily). Six exclusive salt bridges are identified by arrows: R37-E40, E124-K165, K139-E152, E142-K144, R191-E219 and K216-D237. Numbering is based on Est30 (1TQH).</p

Distinctive salt bridge patterns conserved in related lipolytic enzymes.

<p>Structural comparison of the salt bridges content for EstGtA2 (A) LipS (4FBL) (B) and Est30 (1TQH) (C) is shown. At variance with the conserved interloop bridge, the 5 selected salt bridges in EstGtA2 are absent in LipS- and N-related enzymes. Residues exclusive to the N’ subfamily and studied by alanine-scanning mutagenesis are shown in red. Structure of the cap domain is shown in dark gray. The conserved helix of the cap between the three structures is identified. LipS structure share features from N’ and from N subfamilies. The arrow shows salt bridge conserved between EstGtA2 and LipS. The residues forming the interloop salt bridge are shown and identified on the respective structures.</p

Response Monitoring of Acute Lymphoblastic Leukemia Patients Undergoing l‑Asparaginase Therapy: Successes and Challenges Associated with Clinical Sample Analysis in Plasmonic Sensing

Monitoring the response of patients undergoing chemotherapeutic treatments is of great importance to predict remission success, avoid adverse effects and thus, maximize the patients’ quality of life. In the case of leukemia patients treated with E. coli l-asparaginase, monitoring the immune response by the detection of specific antibodies to l-asparaginase in the serum of patients can prevent extended immune response to the drug. Here, we developed a surface plasmon resonance (SPR) biosensor to rapidly detect anti-asparaginase antibodies directly in patients’ sera, without requiring sample pretreatment or dilution. A direct assay with SPR sensing to detect anti-asparaginase antibodies exhibited a limit of detection of 500 pM and a high sensitivity range between 100 nM and 1 μM in pooled and undiluted human serum from a commercial source. While the SPR assay showed excellent reproducibility (12% RSD) in pooled serum, challenges were encountered upon analyzing clinical samples due to high sample-to-sample variability in color and turbidity and, in all likelihood, in composition. As a result, direct detection in clinical samples was unreliable due to factors that may generally affect assays based on plasmonic detection. Addition of a secondary detection step overcame sample variability due to bulk differences in patients’ sera. By those means, the SPR biosensor was successfully applied to the analysis of clinical samples from leukemia patients undergoing asparaginase treatments and the results agreed well with the standard ELISA assay. Monitoring antibodies against drugs is common such that this type of sensing scheme could serve to monitor a plethora of immune responses in sera of patients undergoing treatment