There are a large numbers of protein domains and actually entire

There are a large numbers of protein domains and actually entire proteins, lacking ordered structure below physiological conditions. the chain can be mediated by a triplicate of conserved cytoplasmic sequence motifs, termed Immunoreceptor Tyrosine-centered Activation Motifs (ITAMs), where tyrosine residues are phosphorylated upon TCR engagement within an early and obligatory event in the signaling cascade. We and others possess previously reported that the 13-kDa em /em cyt oplasmic domain ( em /em cyt) can be intrinsically disordered [6-8]. Recently, we’ve discovered that this proteins shows clear symptoms of particular dimerization [7]. Taking into consideration the unstructured character of em /em cyt, the query as to if the proteins continues to Rabbit Polyclonal to HCRTR1 be disordered or undergoes a structural changeover to a folded type in the em /em cyt dimer can be of fundamental importance. According to your previously reported circular dichroic data [7], particular dimerization of em /em cyt will not induce detectable secondary or tertiary framework. Due to the high sensitivity of NMR chemical substance shifts to structural adjustments at atomic quality not really detected with optical strategies, NMR spectroscopy can be unparalleled in its ability to provide detailed structural information on unfolded proteins [9]. ICG-001 inhibition In this study, we use heteronuclear NMR spectroscopy and the 15N,13C-double labeled recombinant em /em cyt protein to monitor the conformational state of em /em cyt on a per-residue level, and prove that dimerization of em /em cyt is not accompanied by a disorder-order transition. Together with our previous results [7], this demonstrates that specific interactions exist between unfolded protein molecules in the em /em cyt homodimer. 2. Materials and methods Unlabeled em /em cyt protein was expressed and purified as previously described [6,7]. Uniformly 15N-labeled and 15N,13C-double labeled cytoplasmic domain of TCR em /em -chain were expressed in 10 L of minimal medium and purified with a combination of ion-exchange and reversed-phase chromatography [7]. All NMR spectra were recorded at 25C in 20 mM phosphate buffer, pH 6.6 on a Varian INOVA spectrometer with 1H Larmor frequencies of 800 and 900 MHz. The diffusion measurements were performed on samples of unlabeled em /em cyt in the protein concentration range 10-500 em /em M using a Varian 600 MHz spectrometer and Stejskal-Tanner pulsed field gradient spin-echo scheme [10] and analysed as described [11]. Experimental errors were estimated as a standard deviation between diffusion coefficients obtained independently for several individual methyl and aromatic peaks. 3. Results and Discussion To characterize em /em cyt homodimerization under solution NMR conditions, we performed NMR diffusion experiments on nonisotopically labeled em /em cyt samples. Pulsed-field gradient NMR techniques were used to measure the translational diffusion coefficients within a protein concentration range of 10 to 500 em /em M. The results are shown in Table 1. The clear dependence of the diffusion coefficients on protein concentration indicates the presence of an oligomeric transition. The em ICG-001 inhibition /em cyt dimer-monomer dissociation constants were estimated based on em D /em t-dependence of molecular weight described elsewhere [10,11] and ranged from 5 to 30 em /em M. This is in a good agreement with the values previously obtained using a variety of other biophysical techniques [7]. Thus, the observed dimerization behavior of em /em cyt under the conditions studied is well suited for NMR characterization of any potential protein folding coupled to dimer formation. Table 1 Concentration dependence of the diffusion coefficients of cyt at 298 K thead th align=”left” rowspan=”1″ colspan=”1″ Protein ( em /em M) /th th align=”center” rowspan=”1″ colspan=”1″ 10 /th th align=”center” rowspan=”1″ colspan=”1″ 20 /th th align=”center” rowspan=”1″ colspan=”1″ 40 /th th align=”center” rowspan=”1″ colspan=”1″ 100 /th th align=”center” rowspan=”1″ colspan=”1″ 500 /th /thead em D /em t* (10-11 m2s-1)10.21 0.569.73 0.259.50 0.239.26 0.238.91 0.22 Open in a separate window *Mean SD. NMR chemical shifts are extremely sensitive to subtle changes in protein conformational ensembles and are particularly useful for determining propensities of secondary structure formation on a residue-by-residue basis in unfolded and partly folded proteins. In this function, we make use of multi-dimensional NMR to characterize the secondary structural propensities of em /em cyt at low and high proteins concentrations where em /em cyt is present predominantly in monomeric and dimeric claims, respectively. The email address details are proven in Fig. 1. Open in another window Fig. 1 Heteronuclear one quantum correlation (HSQC) spectra of em /em cyt at 298 K. (A) The 1H-15N-HSQC spectra of 5 (blue) and 500 (reddish colored) em /em M em /em cyt recorded at 800 MHz. (B) The 1H-13C-HSQC spectra of 5 (blue) and 100 (reddish ICG-001 inhibition colored) em /em M em /em cyt recorded at 900 MHz. The many crowded area on the 1H -13C-HSQC spectra is certainly zoomed at the top. With the dimer-monomer dissociation continuous previously established to be add up to ~10 em /em M (5), the monomer fractions in the 5 em /em M, 100 em /em M, and 500 em /em M em /em cyt samples are.