Postsynchronized analysis

Postsynchronized analysis was performed as reported in previous two papers (Chen

et al., 2012; Chen et al., 2011a) in order to reduce the noise of the data collected. All FRET events were lined up at an identifiable trigger transition and averaged. To get equal durations, all individual traces were extended at the prior and subsequent segment ends. FRET traces were aligned at the beginning (pre-synchronization) or end (post- synchronization). The averaged results revealed the dynamics of the molecules leading up to and following the transition and provided information which was previously hidden due to the noise of the collected data (Fig 2.11). The lifetime of any given state is equal to the reciprocal of transition rate of the given state and the apparent overall translocation rate is the reciprocal of the summed lifetimes of all states in translocation. Two kinetic models were built to study the transition rates by fitting the FRET efficiency. All the results were fitted by Scientist 3.0 (MicroMath) and the detail programs are as follows:

Three-state model 1: Fitted by FRET Define:

56 T, time;

Y1, FRET efficiency of pre-synchronization trace; Y2, FRET efficiency of post-synchronization trace; k1, k2, k3: transition rates;

F1, F2, F3: FRET efficiencies of different complexes;

A, B, C: concentrations of different complexes in pre-synchronization analysis; D, E, F: concentrations of different complexes in post-synchronization analysis. // Simulations IndVars: T DepVars: Y1, Y2 Params: k1, k2, k3, F1, F2, F3 Y1=F1*A+F2*B+F3*C A'=-k1*A B'=k1*A-k2*B C'=k2*B Y2=F3*D+F2*E+F1*F D'=-k3*D E'=k3*D-k2*E F'=k2*E

// Parameter values and constrains //Initial conditions T=0 A=1 B=0 C=0 D=1 E=0 F=0

57 >

Two-state model: Fitted by FRET Define:

T, time;

Y1, FRET efficiency of pre-synchronization trace; Y2, FRET efficiency of post-synchronization trace; k1, k2: transition rates;

F1, F2: FRET efficiencies of different complexes;

A, B: concentrations of different complexes in pre-synchronization analysis; D, E: concentrations of different complexes in post-synchronization analysis. // Simulations IndVars: T DepVars: Y1, Y2 Params: k1, k2, F1, F2 Y1=F1*A+F2*B A'=-k1*A B'=k1*A Y2=F2*D+F1*E D'=-k2*D E'=k2*D

// Parameter values and constrains //Initial conditions

T=0 A=1 B=0 D

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Figure 2.1 Electrophoretic analysis of IF3 by 15% SDS-PAGE gel. Protein concentration measured by Bradford is 180 µM, IF3 (21 KD).

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Figure 2.2 Electrophoretic analysis of His-tagged L11 fractions. Lane 1, L11 (14.5 kDa) after FPLC, 3µL; Lane 2, L11 after FPLC, 9µL; Lanes 3 and 4, L11 pellet debris after centrifugation and before Ni-NTA column purification; Lane 5, 2nd flow through of L11 elution buffer C of Ni-NTA column purification; Lane 6, 1st flow through of L11 elution buffer A of Ni-NTA column purification; Lane 7, 2nd flow through of L11 elution buffer A of Ni-NTA column purification; Lane 8, 1st flow through of L11 elution buffer B; Lane 9, 2nd flow through of L11 elution buffer B of Ni-NTA column purification.

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Figure 2.3Electrophoretic analysis of labeled L11 fractions after FPLC. Lanes 1 - 8 corresponded to FPLC fractions 16 - 23. Fractions 19 - 20 (lanes 4 and 5) were collected and combined.

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Figure 2.4 Electrophoretic analysis of EF-G fractions of FPLC. Lane 1, EF-G (74 kDa) after final concentration by Amicon Ultra ultrafiltration units (after FPLC); Lanes 2 - 8, different FPLC fractions of EF-G; Lane 9, EF-G protein eluted by EF-G elution buffer C after Ni-NTA column and before FPLC purification.

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Figure 2.5 FPLC buffer gradient of EF-G purification. The second red peak (24 - 37 mL) indicated the elution fraction of EF-G protein.

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Figure 2.6 Electrophoretic analysis of labeled EF-G. The labeled product was purified product after FPLC.

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Figure 2.7 Designed EF-G primers

(The mutant sites were marked red).

Primer 1 S453C (32- number of nucleotides)

GG ACT GAC GAA GAA TGCAAC CAG ACC ATC ATC

GAT GAT GGT CTG GTT GCA TTC TTC GTC AGT CC Primer 2 N454C (32)

CT GAC GAA GAA TCT TGC CAG ACC ATC ATC GCG

CGC GAT GAT GGT CTG GCA AGA TTC TTC GTC AG Primer 3 Q455C (32)

GAC GAA GAA TCT AAC TGC ACC ATC ATC GCG GG

CC CGC GAT GAT GGT GCA GTT AGA TTC TTC GTC Primer 4 T456C (33)

GAA GAA TCT AAC CAG TGC ATC ATC GCG GGT ATG

CAT ACC CGC GAT GAT GCA CTG GTT AGA TTC TTC Primer 5 G484C (36)

GTT GAA GCG AAC GTA TGC AAA CCGCAG GTT GCT TAC

GTA AGC AAC CTG CGG TTT GCATAC GTT CGC TTC AAC

Primer 6 K485C (33)

GAA GCG AAC GTA GGT TGC CCGCAG GTT GCT TAC

GTA AGC AAC CTG CGG GCA ACC TAC GTT CGC TTC

Primer Q487C (33)

CG AAC GTA GGT AAA CCG TGC GTT GCT TAC CGT G

C ACG GTA AGC AAC GCA CGG TTT ACC TAC GTT CG

Primer V488C (32)

GTA GGT AAA CCG CAG TGC GCT TAC CGT GAA AC

GT TTC ACG GTA AGC GCA CTG CGG TTT ACC TAC

Primer A489C (33)

GGT AAA CCG CAG GTT TGC TAC CGT GAA ACT ATC

GAT AGT TTC ACG GTAGCA AAC CTG CGG TTT ACC

Primer A690C (32)

CTG AAG TAT GAT GAA TGCCCG AGT AAC GTT GC

GC AAC GTT ACT CGG GCA TTC ATC ATA CTT CAG

Primer S692C (34)

G TAT GAT GAA GCG CCG TGC AAC GTT GCT CAG GCC

65 Primer N693C (31)

GAT GAA GCG CCG AGT TGC GTT GCT CAG GCC G

C GGC CTG AGC AAC GCA ACT CGG CGC TTC ATC

For 231 C-E (36)

GCA GCT GAA GCT TCT GAA GAGCTG ATG GAA AAA TAC

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Figure 2.8 Electrophoretic analysis of PCR product of EF-G derivatives. This gel contains 1% agarose. Lanes 1, 4 and 8, A690C-EF-G DNA with annealing temperatures of 65 °C, 62 °C and 60 °C, respectively; Lanes 2 and 6, S692C-EF-G DNA with annealing temperatures of 65 °C and 62 °C respectively; Lanes 3 and 7, N693C-EF-G DNA with annealing temperatures of 65 °C and 62 °C respectively. Our experimental results confirmed the presence of dsDNA encoding EF-G protein, as it is expected to be 5.2 kilobases in length.

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Figure 2.9 Electrophoretic analysis of wild type EF-Tu. Product was purified by Co(II)-Sepharose resin and the presence of EF-Tu (43.2 kDa) is confirmed by 4-15% SDS-PAGE gel.

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Figure 2.11 Scheme of postsynchronized analysis. All FRET events are lined up at an identifiable trigger transition and averaged. To get equal durations, all individual traces are extended at the prior and subsequent segment ends. FRET traces are aligned at the beginning (pre-synchronization, black) or at the end (post-synchronization, red).

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CHAPTER III EF-G MUTANT SCREENING AND ENSEMBLE