Since HsSMS reaction involves highly charged substrates, it can be expected that electrostatics and potential distribution play significant roles in the HsSMS functions. e) Potential map generation and analysis. The binding energy difference between WT and mutant structures was calculated as follows: where DD G(binding: WT) is the binding energy of the WT monomers and DD G(binding: mutant) is the binding energy of the corresponding mutant monomer chains. The binding energies were calculated for WT structures and all mutant structures (SDmut, CRmut, Pmut and Fmut).
Vmd fieldlines max length free#
Thus, the binding free energy is calculated as: where D G(dimer) is the potential energy of the dimer, D G(C) and D G(D) are the potential energies of monomer ‘‘C’’ and ‘‘D’’ respectively. The energy components include mechanical energy (MM), electrostatic interactions and solvation energy. The binding energy ( DD G(binding)) was defined as the differences between the potential energy of the dimer and the potential energies of the corresponding monomers as described in our previous works. Negative change of binding energy represents that affinity of mutant dimers decreased comparing to that of WT dimers.
Change of binding energy due to mutation corresponds to the change of dimer affinity. With the experience of our previous work, we applied a ‘‘fixed backbone’’ prediction method without a ‘‘backbone pre-relaxation’’ for Eris. In addition, Eris, a webserver based on the Medusa force field, was also used to predict the folding energy change due to these mutations. be mutation independent and cancels out in Eq. (DOCX) Table S3 MSA among 500 homologous proteins. The highly conserved residues are marked with ‘‘*’’ in the alignment. (DOCX) Table S2 The MSA among different species. (DOCX) Table S1 Candidate sites for engineered mutations selected based on 3D structure of HsSMS. The directions of vibrational vectors of the three vibrational modes are shown with orange arrows.
Vmd fieldlines max length code#
The letter code of pathways ‘‘A’’ (the cleft between C-terminal domains) and ‘‘B’’ (the cleft between adjacent C- and N-terminal domains) corresponds to Figure 2 in the main body of the manuscript. The plausible pathways of substrates SPD/SPM are indicated with the black arrows. Figure S1 The first three vibrational modes calculated with the ANM server. The SMS mutant proteins were purified as described above before assays. The entire coding region of both plasmids was verified by DNA sequencing carried out by the Macromolecular Core Facility, Hershey Medical Center.
The following oligodeoxynucleotides (with mism- aches underlined) were used to generate the mutant indicated: 5 9 -ATCACTCTCTGCCAAATTAACATCCCCATCAAGGA TGAGAAT-3 9 (antisense) for S165D 5 9 -TTGGCAGAGAGTGATGAGGCATATCACCGGGCCA TCATG-3 9 (sense) for L175E and T178H 5 9 -GGAGGCATATTGCGTGAAATAGTCAAA-3 9 (sense) for C206R 5 9 -TTTGACTATTTCACGCAATATGCCTCC-3 9 (antisense) for C206R The entire coding sequence of each of the SMS mutants was verified by DNA sequencing to ensure that no other mutations were introduced during PCR. The four amino acid mutated HsSMS was generated by PCR and subcloned into the pQE30 vector. All assays were carried out in triplicate, and results agreed within 6 5%. Reactions were run with an amount of enzyme that gave a linear rate of MTA production over the assay time period. All assays were conducted with an amount of protein and for a period of time in which product formation was linear with time. The reaction was stopped by acidification and MTA separated from dcAdoMet using phosphocellulose columns. Activity was measured by following the production of MTA from dcAdoMet in 100 mM sodium phosphate buffer (pH 7.5), in the presence of SPD.
Recombinant human SS was purified by immobilized metal affinity chromatography using TALON affinity resin (Clontech Laboratories, Palo Alto, CA, U.S.A.), in accordance with manufacturer’s instruction. The resulting plasmids were used to transform XL1- Blue cells. A DNA fragment encoding HsSMS was amplified by PCR and subcloned into the pQE-30 vector downstream of the polyhistidine coding region. This isĪchieved by placing the magnetic field on an R-phi-Z mesh andĬonstructing splines over that mesh. The FIELDLINES code follows fieldlines in a toroidal domain. Parallelized over the field line trajectories. Vacuum, VMEC, PIES, or SPEC equilibria, and is The FIELDLINES code follows field lines in a toroidal domain given a
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