NMR spectroscopy with proteins based on observation of a small number of spins with outstanding spectral properties, which either may be present naturally or introduced by techniques such as site-specific isotope labeling, yielded biologically relevant information on human hemoglobin (M ϭ 65,000) as early as 1969 (1), and subsequently also for significantly larger systems such as Igs (2). In contrast, the use of NMR for de novo structure determination (3, 4) so far has been limited to relatively small molecular sizes, with the largest NMR structure below molecular weight 30,000. Although NMR in structural biology may, for practical reasons of coordinated use with x-ray crystallography (5), focus on smaller molecular sizes also in the future, considerable effort goes into attempts to extend the size limit to bigger molecules (for example, see refs. 6-8). Here we introduce transverse relaxation-optimized spectroscopy (TROSY) and present experimental data and theoretical considerations showing that this approach is capable of significantly reducing transverse relaxation rates and thus overcomes a key obstacle opposing solution NMR of larger molecules (7).At the high magnetic fields typically used for studies of proteins and nucleic acids, chemical shift anisotropy interaction (CSA) of 1 H, 15 N, and 13 C nuclei forms a significant source of relaxation in proteins and nucleic acids, in addition to dipole-dipole (DD) relaxation. This leads to increase of the overall transverse relaxation rates with increasing polarizing magnetic field, B 0 . Nonetheless, transverse relaxation of amide protons in larger proteins at high fields has been reduced successfully by complete or partial replacement of the nonlabile hydrogen atoms with deuterons and, for example, more than 90% of the 15 N, 13 C ␣ , and 1 H N chemical shifts thus were assigned in the polypeptide chains of a protein-DNA complex of size 64,000 (6). TROSY uses spectroscopic means to further reduce T 2 relaxation based on the fact that cross-correlated relaxation caused by DD and CSA interference gives rise to different relaxation rates of the individual multiplet components in a system of two coupled spins 1 ⁄2, I and S, such as the 15 N-1 H fragment of a peptide bond (9, 10). Theory shows that at 1 H frequencies near 1 GHz nearly complete cancellation of all transverse relaxation effects within a 15 N-1 H moiety can be achieved for one of the four multiplet components. TROSY observes exclusively this narrow component, for which the residual linewidth is then almost entirely because of DD interactions with remote hydrogen atoms in the protein. These can be efficiently suppressed by 2 H-labeling, so that in TROSY-type experiments the accessible molecular size for solution NMR studies no longer is primarily limited by T 2 relaxation. TheoryWe consider a system of two scalar coupled spins 1 ⁄2, I and S, with a scalar coupling constant J IS , which is located in a protein molecule. T 2 relaxation of this spin system is dominated by the DD coupling of I and S a...

The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90 -230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered ''tail.'' The globular domain contains three ␣-helices comprising the residues 144 -154, 173-194, and 200 -228 and a short anti-parallel ␤-sheet comprising the residues 128 -131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219 -228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219 -226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions. P rion proteins (PrP) are associated with transmissible spongiform encephalopathies (TSE), which are invariably fatal diseases characterized by loss of motor control, dementia, and paralysis wasting (1, 2). Human TSEs include Creutzfeldt-Jakob disease, fatal familial insomnia, the Gerstmann-Sträussler-Scheinker syndrome, and kuru, and there is bovine spongiform encephalopathy in cattle and scrapie in sheep. The ''proteinonly'' hypothesis (3, 4) proposes that TSEs are caused by the conversion of a ubiquitous ''cellular form'' of PrP (PrP C ) into an aggregated ''scrapie form'' (PrP Sc ). According to this model, the prion protein (PrP) would at the same time be target and infectious agent in TSEs, which could explain that this class of diseases can be traced to infectious, inherited, and spontaneous origins (2, 5). PrP Sc is characterized by a high ␤-sheet content, insolubility in detergents, and resistance to proteolysis in its aggregated form (6-8) whereas PrP C is a soluble protein with a high content of ␣-helices (8, 9) and high susceptibility to proteolytic digestion. No chemical modifications have as yet been identified by which the two PrP forms would differ (10).Considering that the protein-only hypothesis suggests a change of protein conformation as a possible cause of the onset of TSEs, the three-dimensional prion protein structures have attracted keen interest. So far, nuclear magnetic resonance (NMR) solution studies have been described for monomeric, cellular forms of PrP of the two most widely used laboratory animals in prion research, the mouse (m) and the Syrian hamster (sh)...

The 'protein only' hypothesis states that a modified form of normal prion protein triggers infectious neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), or Creutzfeldt-Jakob disease (CJD) in humans. Prion proteins are thought to exist in two different conformations: the 'benign' PrPcform, and the infectious 'scrapie form', PrPsc. Knowledge of the three-dimensional structure of PrPc is essential for understanding the transition to PrPsc. The nuclear magnetic resonance (NMR) structure of the autonomously folding PrP domain comprising residues 121-231 (ref. 6) contains a two-stranded antiparallel beta-sheet and three alpha-helices. This domain contains most of the point-mutation sites that have been linked, in human PrP, to the occurrence of familial prion diseases. The NMR structure shows that these mutations occur within, or directly adjacent to, regular secondary structures. The presence of a beta-sheet in PrP(121-231) is in contrast with model predictions of an all-helical structure of PrPc (ref. 8), and may be important for the initiation of the transition from PrPc to PrPsc.

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.