Gerhard Wider

NMR characterization of the full-length recombinant murine prion protein, mPrP(23–231)

The recombinant murine prion protein, mPrP(23–231), was expressed in E. coli with uniform 15N‐labeling. NMR experiments showed that the previously determined globular three‐dimensional structure of the C‐terminal domain mPrP(121–231) is preserved in the intact protein, and that the N‐terminal polypeptide segment 23–120 is flexibly disordered. This structural information is based on nearly complete sequence‐specific assignments for the backbone amide nitrogens, amide protons and α‐protons of the polypeptide segment of residues 121–231 in mPrP(23–231). Coincidence of corresponding sequential and medium‐range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121–231) are also present in mPrP(23–231), and near‐identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three‐dimensional fold of mPrP(121–231) is also preserved in the intact protein. The linewidths in heteronuclear 1H–15N correlation spectra and 15N{1H}‐NOEs showed that the well structured residues 126–230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23–231) outside of this domain.

Ubiquitin-Binding Domains in Y-Family Polymerases Regulate Translesion Synthesis

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 polη and polι to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of polη 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.

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

Abstract A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.

Homonuclear two-dimensional 1H NMR of proteins. Experimental procedures

Experimental techniques used for homonuclear 2D 1H NMR studies of proteins are described. A brief survey of the general strategy for structural studies of proteins by 2D NMR is included. The main part of the paper discusses guidelines for the selection of experimental techniques, the elimination of artifacts and unwanted peaks in protein 2D 1H NMR spectra, suppression of the solvent line in H2O solutions, experimental parameters, numerical data processing before and after Fourier transformation, and suitable presentations of complex 2D NMR spectra.

Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance

A determination of the spatial structure of the polypeptide hormone glucagon bound to perdeuterated dodecylphosphocholine micelles is described. A map of distance constraints between individually assigned hydrogen atoms of the polypeptide chain was obtained from two-dimensional nuclear Overhauser enhancement spectroscopy. These data were used as the input for a distance geometry algorithm for computing conformations that would be compatible with the experiments. In the region from residues 5 to 29 the mobility of the polypeptide backbone and most of the amino acid side-chains was found to be essentially restricted to the overall rotational tumbling of the micelles. The secondary structure in this region includes three turns of irregular alpha-helix in the segment of residues 17 to 29 near the C terminus, a stretch of extended polypeptide chain from residues 14 to 17, an alpha-helix-like turn formed by the residues 10 to 14 and another extended region from residues 5 to 10. In the N-terminal tetrapeptide H-His-Ser-Gln-Gly- the two terminal residues are highly mobile, indicating that they extend into the aqueous phase, and the mobility of the residues Gln3 and Gly4 appears to be only partially restricted by the binding to the micelle. The absence of long range nuclear Overhauser effects between the peptide segments 5-9 and 11-29, and between 5-16 and 19-29 shows that the polypeptide chain does not fold back on itself and hence that micelle-bound glucagon does not adopt a globular tertiary structure. Previously it was shown that the polypeptide backbone of glucagon is located close to and runs roughly parallel to the micelle surface. Combination of these observations suggests that the overall spatial arrangement of the glucagon polypeptide chain in a lipid-water interphase is largely determined by the topology of the lipid support, in the present case the curvature of the dodecylphosphocholine micelles. The tertiary structure is further characterized by the formation of two hydrophobic patches by the side-chains of Phe6, Tyr10 and Leu14, and the side-chains of Ala19, Phe22, Val23, Trp25 and Leu26, respectively.

Processing of multi dimensional NMR data with the new software PROSA

SummaryThe new program PROSA is an efficient implementation of the common data-processing steps for multi-dimensional NMR spectra. PROSA performs linear prediction, digital filtering, Fourier transformation, automatic phase correction, and baseline correction. High efficiency is achieved by avoiding disk storage of intermediate data and by the absence of any graphics display, which enables calculation in the batch mode and facilitates porting PROSA on a variety of different computer systems; including supercomputers. Furthermore, all time-consuming routines are completely vectorized. The elimination of a graphics display was made possible by the use of a new, reliable automatic phase-correction routine. CPU times for complete processing of a typical heteronuclear three-dimensional NMR data set of a protein vary between less than 1 min on a NEC SX3 supercomputer and 40 min on a Sun-4 computer system.

Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules

In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings [insensitive nuclei enhanced by polarization transfer (INEPT)] are key elements of the pulse schemes. For molecular weights beyond 100,000, transverse relaxation during the transfer time may become a limiting factor. This paper presents a transfer technique for work with big molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which largely reduces the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. As a first implementation, [15N,1H]-correlation experiments were designed that make use of cross-correlation between dipole-dipole coupling and chemical shift anisotropy of the 15N---1H-moieties for both CRINEPT and transverse relaxation-optimized spectroscopy (TROSY). When compared with INEPT-based [15N,1H]-TROSY, these experiments yielded up to 3-fold signal enhancement for amide groups of a 110,000-Da protein in aqueous solution at 4 degrees C. CRINEPT opens avenues for solution NMR with supramolecular structures such as membrane proteins solubilized in micelles or lipid vesicles, proteins attached to nucleic acid fragments, or oligomeric proteins.

Automated projection spectroscopy (APSY).

This work presents the automated projection spectroscopy (APSY) method for the recording of discrete sets of j projections from N-dimensional (N > or = 3) NMR experiments at operator-selected projection angles and automatic identification of the correlation cross peaks. The result from APSY is the fully automated generation of the complete or nearly complete peak list for the N-dimensional NMR spectrum from a geometric analysis of the j experimentally recorded, low-dimensional projections. In the present implementation of APSY, two-dimensional projections of the N-dimensional spectrum are recorded by using techniques developed for projection-reconstruction spectroscopy [Kupce,E.& Freeman, R. (2004) J. Am. Chem. Soc. 126, 6429-6440]. All projections are peak-picked with the available automated routine atnos. The previously undescribed algorithm GAPRO (geometric analysis of projections) uses vector algebra to identify subgroups of peaks in different projections that arise from the same resonance in the N-dimensional spectrum, and from these subgroups it calculates the peak positions in the N-dimensional frequency space. Unambiguous identification thus can be achieved for all cross peaks that are not overlapped with other peaks in at least one of the N dimensions. Because of the correlation between the positions of corresponding peaks in multiple projections, uncorrelated noise is efficiently suppressed, so that APSY should be quite widely applicable for correlation spectra of biological macromolecules, which have intrinsically low peak density in the N-dimensional spectral space.

Recombinant full‐length murine prion protein, mPrP(23–231): purification and spectroscopic characterization

The cellular prion protein of the mouse, mPrPC, consists of 208 amino acids (residues 23–231). It contains a carboxy‐terminal domain, mPrP(121–231), which represents an autonomous folding unit with three α‐helices and a two‐stranded antiparallel β‐sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23–231), in the cytoplasm of Escherichia coli. mPrP(23–231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques. Comparison of near‐UV circular dichroism, fluorescence and one‐dimensional 1H‐NMR spectra of mPrP(23–231) and mPrP(121–231) shows that the amino‐terminal segment 23–120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three‐dimensional structure as detected by these techniques, indicating that the residues 121–231 might be the only polypeptide segment of PrPC with a defined three‐dimensional structure.

Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY

AbstractThis paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a