NMR Studies of PolyQ-terminated Huntingtin

NMR Studies of PolyQ-terminated Huntingtin Peptides In Vitro and in Brain Extracts
Pieter E. S. Smith1; Maria Baias1; Koning Shen2; Lukasz Joachimiak2; Judith Frydman2; Lucio Frydman1
1
Weizmann Institute of Science, Rehovot, Israel; 2Stanford University, Stanford, CA
Studying proteins at atomic resolution both in vitro and in their native environments, is fundamental to understanding
protein folding and aggregation. This work studies a CAG expansion within the huntingtin (Htt) gene, that encodes a
polymorphic glutamine tract at the N-terminus of the protein and that is associated with Huntington's disease. Although Htt
peptide fragments naturally occur in the brain environment, their function is not well understood. 1 To help elucidate the
molecular basis of Htt aggregation, we investigate wild type Htt with a 17 residue polyQ stretch (HttQ17). Studying Htt
peptides presents a number of unique challenges: they display a high degree of conformational flexibility leading to
averaging of NMR chemical shifts, and a large portion of their backbones are solvent-exposed leading to fast hydrogen
exchange and causing extensive line broadening. In vitro, we suppress hydrogen exchange by dissolving HttQ17 in a low
pH solution (Fig 1a). Resonances in the neutral (pH = 7.4) in vitro samples were then mapped to their low pH counterparts
by performing an NMR titration (Fig 1b). These data further reveal structural changes associated with changes in acidic
and basic HttQ17 residues' ionization states. The changes we observe associated with peptide dynamics and hydrogen
exchange seem to be largely reversible (Fig 2a), suggesting that temperature associated changes in peptide dynamics
could be probed in a similar manner. T1, T2, and 1H-15N heteronuclear NOE experiments are used to extract order
parameters for the Htt peptide aggregates under in vitro conditions. Using the key resonances we assigned in the spectra
of Htt peptides under neutral in vitro conditions allowed us to identify conformational changes and/or post-translation
modifications in a in murine brain extract and Htt mixture (Fig 2b). These hydrodynamic radii are compared with those
obtained for Htt peptides' monomeric forms, which are observed under low pH in vitro conditions. Additional structural
information is sought on Htt and brain extract aggregates after sedimentation using solid-state NMR. The detailed
knowledge gained from these studies will be relevant to better understand the aggregation of Htt. The approach utilized to
assign Htt peptide resonances, based on assignments at low pH where H-exchange is suppressed and using a pH
titration to map chemical shifts changes from low pH to neutral pH, will likely be useful in investigations of other
intrinsically disordered peptides as well.
Fig 1. (a) A 1H-15N band-selective excitation optimized-flip-angle short-transient (BEST)
HSQC of HttQ17 in 10 % aqueous formic acid. Assignments were obtained utilizing BEST
HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, and HN(CO)CACB experiments
(HttQ17's amino acid sequence is shown above the spectrum). (b) A pH titration experiment
performed on HttQ17 in 50 mM phosphate buffer (pH is indicated). The pH of the NMR
sample was measured between spectrum acquisitions using a pH micro-electrode. An
illustrative mapping of the E5's chemical shift from low pH (1.75) to neutral pH (7.08) is
indicated.
Fig
2.
(a)
Experiments
show
temperature associated changes in
NMR spectra are reversible: 4 °C before
heating to 37 °C (top), and after
(bottom).
(b)
Monitoring
HttQ17
aggregation in murine brain extract with
1
H-15N BEST HSQC .
Acknowledgments
We thank Drs. Martin S. Nausner and Tali Scherf for help in setting up NMR experiments and useful discussion. We thank
Dr. Martin S. Nausner for help in preparing samples.
References
1. The Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is
expanded and unstable on Huntington's disease chromosomes. Cell 72(6): 971-983.

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