Preliminary characterization of LacI structure and function by NMR

In order to gain a more detailed structural view of the recombinant products, NMR spectroscopy of isotopically labeled LacI and its constituent domains was performed. The simplest and most informative NMR experiment at this stage is 1H-15N heteronuclear single quantum coherence (HSQC). This spectrum is often referred to as the “fingerprint” of a protein because it yields a resonance for each amide H-N bond vector. However, the identity or “assignment” of each resonance cannot be determined without additional data collection. At the time of this study, resonance assignments were only available for the isolated DBD bound to Osym

(via Prof. Rob Kaptein’s group deposited in the Biological Magnetic Resonance Bank (BMRB)). However, even without assignments, HSQC spectra can still be interpreted with respect to structure and function.

sensitive to changes in the local chemical environment. As such, it is an extremely valuable readout for conformational changes that may accompany binding events. The functionality of a protein can therefore be determined simply by visual inspection of chemical shift perturbations. With respect to LacI, we are interested in probing IPTG-binding and operator-binding.

Figure 2.121H-15N TROSY-HSQC spectra of LacI and LacI RD apo and IPTG-bound states (A) Apo LacI. (B) LacI bound to IPTG. (C) Apo RD. (D) RD bound to IPTG. All data were collected at 750 MHz and 25˚C. Negative peaks are shown in red.

As described in Chapter 1, the operator we have chosen for this study is Osym because

the perfectly symmetric DNA sequence simplifies NMR spectra. Moreover, the interaction between LacI and Osym has been characterized in the bulk of structural studies performed to date.

Both IPTG and Osym bind LacI with sub-micromolar dissociation constants. High affinity binding is

exchange between free and bound states is slower than the difference between the free and bound resonance frequencies. In other words, apo and bound states yield unique peaks rather than one peak with a population-averaged frequency. This is beneficial at this preliminary stage of characterization because it allows for the rapid detection of non-functional or under-titrated samples.

Figure 2.13 1H-15N TROSY-HSQC spectra of LacI operator-bound states (A) LacI-Osym binary complex. (B) LacI-Osym-IPTG ternary complex. All data were collected at 750 MHZ and 35˚C. Negative peaks are shown in red.

Figure 2.12 shows 1H-15N TROSY HSQC spectra for apo and IPTG-bound LacI (2.12a-b, respectively) and LacI RD (2.12c-d, respectively) collected at 25˚C. The temperature was chosen in accordance with previously reported thermal denaturation melting temperatures of dimeric LacI. The LacI RD and DBD have different melting temperatures: 55˚C (measured by us using fluorescence emission, data not shown) and 37˚C [177], respectively. As such, 25˚C was chosen to accommodate the less thermostable DBD. The dispersion of chemical shifts indicates that the

proteins are well folded. A single set of resonances is observed, which is to be expected for a symmetric dimer. Importantly, drastic changes in chemical shift are observed upon IPTG-binding which confirms that the proteins are functional. Qualitatively, it is clear that spectra of the RD are highly similar to those of LacI (to be discussed further in Chapter 3) which suggests that the constituent domains will be useful for chemical shift assignment.

Spectra of LacI bound to a duplexed 22-base pair Osym are shown in Figure 2.13. The 1H-

15_{N TROSY HSQC spectrum of the LacI-O}

sym binary complex is shown in 2.13a and the 1H-15N

TROSY HSQC spectrum of the LacI-Osym-IPTG ternary complex is shown if 2.13b. Both were

collected at 35˚C to improve molecular tumbling. To our knowledge, thermal denaturation studies have not been reported for the dimeric LacI-Osym binary or LacI-Osym-IPTG ternary complex.

However, such studies have been reported for tetrameric LacI bound to non-operator DNA and indicate that DNA-binding increases the melting temperature of LacI [178]. Curiously, it was shown that IPTG-binding has no affect on the melting temperature of LacI, whether free or bound to DNA. As a control, spectra were also collected at 25˚C and no significant differences were observed (data not shown). The spectral quality is excellent. Again, both states yield spectra with a single set of resonances, consistent with a symmetric dimer. Importantly, we present (to our knowledge) the first high resolution view of the ternary LacI-Osym-IPTG complex which is

recalcitrant to crystallization.

Figure 2.141H-15N HSQC spectra of the isolated LacI DBD (A) Apo DBD at pH = 4.5. (B) Apo DBD at pH = 7.4. (C) DBD-Osym complex. (A-B) were collected at 500MHz and 25˚C whereas (C)

was collected at 500 MHz and 35˚C. All data were processed identically.

In order to assess the quality of the recombinant LacI DBD produced using the pET- 15b/BL21(DE3) expression system, 1H-15N HSQC spectra were collected at 25˚C. Initial NMR studies were performed at the same pH used in studies of LacI and LacI RD, pH = 7.4. However, it was apparent that many resonances were broadened beyond detection. While line broadening can arise for a variety of reasons, one plausible explanation is accelerated HX. In order to test this, a 1H-15N HSQC spectrum was also collected at pH = 4.5. Figure 2.14a-b shows spectra of the apo DBD at pH = 4.5 and pH = 7.4, respectively. It is clear that the peaks that were not observed at pH = 7.4 are observable at pH = 4.5.

In order to test the functionality of the isolated DBD, a 1H-15N HSQC spectrum of the DBD were collected in the presence of saturating amounts of Osym as shown in Figure 2.14c. The dramatic changes in chemical shift indicate that the isolated DBD is competent to bind Osym, thus confirming its functionality.