In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein ␣-synuclein (␣S) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, ␣S to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric ␣S assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to microseconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and͞or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of ␣S constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.is the second most common neurodegenerative disease and the most common movement disorder, affecting 1-2% of the population over 65 years of age (1). The cause of PD is as yet unclear due in part to a complex etiology involving a combination of genetic susceptibility and numerous environmental factors (2). Proteinaceous aggregates in motor neurons of the substantia nigra and locus coeruleus are characteristic of idiopathic PD. An abundant component of these so-called Lewy bodies is the presynaptic protein ␣-synuclein (␣S) (3). Three genetic mutations in ␣S (A30P, E46K and A53T) have been identified in autosomal-dominantly inherited early-onset PD (4, 5). In vitro, different conditions such as increased temperature, lower pH, and naturally occurring polyamines accelerate ␣S aggregation (6, 7). Compelling evidence now supports a cytotoxic role in PD for protofibrils, early oligomers of ␣S (8).In other amyloid-related neurological disorders, such as Creutzfeldt-Jakob disease, protein oligomerization͞aggregation requires destabilization of a soluble monomeric protein followed by the formation of highly ordered, ␤-sheet-like fibrillar structures (9). ␣S, however, belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD, Fourier transform IR, or NMR spectroscopy (6, 10, 11), although recent evidence indicates the existence of distinct, functionally relevant intramolecular interactions (refs. 7 and 12 and this work). The challenge is to rationalize in structural terms the inactive state of the soluble, unstructured protein and the potentiation of aggregation by point mutations, ligand binding, or changes in solution conditions. During the past decade numerous NMR techniques have been developed for elucidating the unfolded states of proteins in atomic detail (13). 15 N-relaxation time measurements and paramagnetic relaxation enhancement (PRE) from site-directed spin labeling allo...

MARS a program for robust automatic backbone assignment of 13 C/ 15 N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only 13 C α / 13 C β connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment.

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.