Nascent structure in the kinase anchoring domain of microtubule-associated protein 2

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Abstract

Biological processes are often viewed as highly ordered interactions between well-folded protein domains. The specific interactions exhibited by certain highly abundant neuronal proteins such as microtubule-associated protein 2 (MAP2) and tau stand in stark contrast because these proteins do not show evidence of structure by standard biophysical assays, yet they do bind to specific targets. It is conceivable that there are regions of MAP2 and tau with propensity to form structural domains upon binding a target. To search for evidence of such regions, limited proteolysis experiments were carried out on MAP2c, the smallest MAP2 isoform. Increased protease resistance was observed around the binding site for the RII subunit of cAMP-dependent protein kinase. Protein constructs spanning this region were produced based on the long-lived tryptic fragments Ser44–Arg93 and Ile94–Arg182, and were probed for structure using spectroscopic methods. The results support the existence of regions of nascent structure in the N-terminal region of MAP2c, which are believed to contribute to its regulatory function.

Section snippets

Experimental procedures

Protein expression and purification. Full-length rat MAP2c was expressed as described previously [16]. The lysate was boiled for 10 min, swirling periodically [17], and cleared by centrifugation for 20 min at 10,000g. MAP2c was further purified on a 1 ml Pharmacia HiTrap SP cation exchange column using a NaCl gradient (0–500 mM) in 50 mM NaAc buffer at pH 7.0 containing 1 mM EDTA and 1 mM EGTA.

The genes for the N-terminal MAP2c fragments were amplified by PCR from the full-length rat MAP2c gene and

Limited proteolysis

To search for the presence of specific folded units in MAP2, the smallest MAP2 isoform (MAP2c, 467 amino acids; Fig. 1) was subjected to limited tryptic and chymotryptic digestions, respectively (Fig. 2). Two specific fragments that were more resistant to tryptic digestion than the rest of the protein were identified (Fig. 2, Fig. 3). After separation by HPLC, MALDI-TOF mass spectrometry showed these to be consecutive fragments Ser44–Arg93 and Ile94–Arg182 (Figs. 2B and C). The observation of a

Discussion

The current consensus view of biological processes involves interactions between well-folded proteins or protein domains in a highly ordered manner. In contrast, over the past few years a growing number of studies of physiologically active proteins show that they are “unfolded” in vitro, e.g., [30]. Many of these proteins are not completely unfolded in the traditional sense of a random coil, but rather exist in dynamic equilibrium between a set of preferred conformations [31].

The

Acknowledgements

We thank members of the Halpain and Chazin labs for assistance in the early stages of this project, and Rachel Ozer, Laura Mizoue, and Robley Williams for helpful discussions. Jonathan Sheehan is acknowledged for remote information processing. This work was supported by operating Grants from the National Institutes of Health (RO1 MH50861 to S.H.; RO1 GM 40120 and GM62112 to W.J.C.) and a postdoctoral fellowship to A.M. from the Swedish Foundation for International Cooperation in Research and

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    Abbreviations: AKAP, A-kinase anchoring protein; CD, circular dichroism; MALDI, matrix-assisted laser desorption ionization, MAP2, microtubule-associated protein 2; MT, microtubule; NMR, nuclear magnetic resonance; RII, regulatory domain of cAMP-dependent protein kinase type II; TMAO, trimethylamine N-oxide; TFE, trifluoroethanol.

    1

    Present address: Laboratory for Biomolecular NMR Spectroscopy, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.

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