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Thomas Szyperski

Publications and source records attributed to Thomas Szyperski.

At least 19 recordsLinked to original sources
PubMed2007

J-GFT NMR for precise measurement of mutually correlated nuclear spin-spin couplings.

G-matrix Fourier transform (GFT) NMR spectroscopy is presented for accurate and precise measurement of chemical shifts and nuclear spin-spin couplings correlated according to spin system. The new approach, named "J-GFT NMR", is based on a largely extended GFT NMR formalism and promises to have a broad impact on projection NMR spectroscopy. Specifically, constant-time J-GFT (6,2)D (HA-CA-CO)-N-HN was implemented for simultaneous measurement of five mutually correlated NMR parameters, that is, 15N backbone chemical shifts and the four one-bond spin-spin couplings 13Calpha-1Halpha, 13Calpha-13C', 15N-13C', and 15N-1HNu. The experiment was applied for measuring residual dipolar couplings (RDCs) in an 8 kDa protein Z-domain aligned with Pf1 phages. Comparison with RDC values extracted from conventional NMR experiments reveals that RDCs are measured with high precision and accuracy, which is attributable to the facts that (i) the use of constant time evolution ensures that signals do not broaden whenever multiple RDCs are jointly measured in a single dimension and (ii) RDCs are multiply encoded in the multiplets arising from the joint sampling. This corresponds to measuring the couplings multiple times in a statistically independent manner. A key feature of J-GFT NMR, i.e., the correlation of couplings according to spin systems without reference to sequential resonance assignments, promises to be particularly valuable for rapid identification of backbone conformation and classification of protein fold families on the basis of statistical analysis of dipolar couplings.

Carbon Isotopes↗
PubMed2007

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates.

The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39x10(-5) s-1 and 4.44x10(-5) s-1) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

Culture Media↗
PubMed2006

Principles and applications of GFT projection NMR spectroscopy.

The two defining features of G-matrix Fourier transform (GFT) projection NMR spectroscopy are (i) repeated joint sampling of several indirect chemical shift evolution periods of a multidimensional NMR experiment so that transfer amplitudes are generated which are proportional to all possible permutations of cosine and sine modulations of the individual shifts, and (ii) linear combination of the subspectra resulting from such repeated joint sampling in the time or frequency domain which yields edited subspectra containing signals encoding phase-sensitively detected linear combinations of the jointly sampled shifts. This review sketches the underlying principles of GFT NMR and outlines its relation to further developments such as the reconstruction of multidimensional NMR spectra.

Fourier Analysis↗
PubMed2005

Probing structure and functional dynamics of (large) proteins with aromatic rings: L-GFT-TROSY (4,3)D HCCH NMR spectroscopy.

NMR assignment of aromatic rings in proteins is a prerequisite for obtaining high-quality solution structures of proteins and for studying the dynamics and folding of their molecular cores. Here we present sensitive PFG-PEP L-GFT-(TROSY) (4,3)D HCCH NMR for identification of aromatic spin systems based on four-dimensional (4D) spectral information which can be rapidly obtained with high digital resolution. The G-matrix Fourier Transform (GFT) experiment relies on newly introduced longitudinal relaxation (L-)optimization for aromatic protons and is optimally suited for both sensitivity and sampling limited data collection, making it particularly attractive for NMR-based structural genomics. Applications are presented for 21 and 13 kDa proteins HR41 and MaR11, targets of the Northeast Structural Genomics Consortium for which data collection is, respectively, sensitivity and sampling limited. Complete assignment of aromatic rings enabled high-quality NMR structure determination, and nearly complete analysis of aromatic proton line widths allowed one to assess the flipping of most rings in HR41. Specifically, the ring of Tyr90 flips very slowly on the seconds time scale, thereby proving the absence of fast larger-amplitude motional modes which could allow the ring to flip. This indicates remarkable rigidity of the substructure in which the ring is embedded. Tyr90 is conserved among ubiquitin-conjugating enzymes E2, to which HR41 belongs, and is located in spatial proximity to the interface between E2 and ubiquitin protein ligase E3. Hence, the conformational rigidity and/or the slow motional mode probed by the ring might be of functional importance.

Carbon Isotopes↗
PubMed2005

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of deltaC(p) of protein unfolding.

Cold and heat denaturation of the double mutant Arg 3-->Glu/Leu 66-->Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D (1)H NMR spectroscopy in the temperature range from -12 degrees C in supercooled water up to +70 degrees C. The fraction of unfolded protein, f (u), was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be -11 degrees C and 39 degrees C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, DeltaC(p) of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.

Algorithms↗
PubMed2005

A structure-based model of the c-Myc/Bin1 protein interaction shows alternative splicing of Bin1 and c-Myc phosphorylation are key binding determinants.

The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.

Adaptor Proteins, Signal Transducing↗
PubMed2005

NMR data collection and analysis protocol for high-throughput protein structure determination.

A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.

Data Collection↗
PubMed2005

G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination.

A protocol for high-quality structure determination based on G-matrix Fourier transform (GFT) NMR is presented. Five through-bond chemical shift correlation experiments providing 4D and 5D spectral information at high digital resolution are performed for resonance assignment. These are combined with a newly implemented (4,3)D GFT NOESY experiment which encodes information of 4D 15N/15N-, 13C(alipahtic)/15N-, and 13C(aliphatic)/13C(aliphatic)-resolved [1H,1H]-NOESY in two subspectra, each containing one component of chemical shift doublets arising from 4D --> 3D projection at omega1:Omega(1H) +/- Omega(X) [X = 15N,13C(aliphatic)]. The peaks located at the centers of the doublets are obtained from simultaneous 3D 15N/13C(aliphatic)/13C(aromatic)-resolved [1H,1H]-NOESY, wherein NOEs detected on aromatic protons are also obtained. The protocol was applied for determining a high-quality structure of the 14 kDa Northeast Structural Genomics consortium target protein, YqfB (PDB ID ). Through-bond correlation and NOESY spectra were acquired, respectively, in 16.9 and 39 h (30 h for shift doublets, 9 h for central peaks) on a 600 MHz spectrometer equipped with a cryogenic probe. The rapidly collected highly resolved 4D NOESY information allows one to assign the majority of NOEs directly from chemical shifts, which yields accurate initial structures "within" approximately 2 angstroms of the final structure. Information theoretical "QUEEN" analysis of initial distance limit constraint networks revealed that, in contrast to structure-based protocols, such NOE assignment is not biased toward identifying additional constraints that tend to be redundant with respect to the available constraint network. The protocol enables rapid NMR data collection for robust high-quality structure determination of proteins up to approximately 20-25 kDa in high-throughput.

Bacterial Proteins↗
PubMed2005

Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G2FT NMR experiments.

A suite of novel (5,3)D G2FT triple resonance NMR experiments encoding highly resolved 5D spectral information is presented for sequential resonance assignment of proteins exhibiting high chemical shift degeneracy. Efficient resonance assignment is achieved by separate joint sampling of (i) chemical shifts which solely serve to provide increased resolution and (ii) shifts which also provide sequential connectivities. In these G2FT experiments, two G-matrix transformations are employed. Peaks are resolved along a first GFT dimension at both Omega(15N) + Omega(13C') and Omega(15N) - Omega(13C'), or at Omega(15N) + Omega(13Calpha) and Omega(15N) - Omega(13Calpha), to break backbone 15N,1HN chemical shift degeneracy. Sequential connectivities are established along a second GFT dimension by measuring intraresidue and sequential correlations at 2Omega(13Calpha), Omega(13Calpha + 13Cbeta), and Omega(13Calpha - 13Cbeta), or at Omega(13Calpha + 1Halpha) and Omega(13Calpha - 1Halpha), to resolve 13Calpha/beta,1Halpha chemical shift degeneracy. It is demonstrated that longitudinal proton relaxation optimization of out-and-back implementations suitable for deuterated proteins and nonlinear data sampling combined with maximum entropy reconstruction further accelerate G2FT NMR data acquisition speed. As a result, the spectral information can be obtained within hours, so that (5,3)D G2FT experiments are viable options for high-throughput structure determination in structural genomics. Applications are presented for 17 kDa alpha-helical protein YqbG and 13.5 kDa protein rps24e, targets of the Northeast Structural Genomics consortium, as well as for 9 kDa protein Z-domain. The high resolving power of the G2FT NMR experiments makes them attractive choices to study alpha-helical globular/membrane or (partially) unfolded proteins, thus promising to pave the way for NMR-based structural genomics of membrane proteins.

Fourier Analysis↗
PubMed2005

NMR solution structure of Thermotoga maritima protein TM1509 reveals a Zn-metalloprotease-like tertiary structure.

The 150-residue protein TM1509 is encoded in gene YF09_THEMA of Thermotoga maritima. TM1509 has so far no functional annotation and belongs to protein family UPF0054 (PFAM accession number: PF02130) which contains at least 146 members. The NMR structure of TM1509 reveals an alpha+beta fold comprising a four stranded beta-sheet with topology A( upward arrow), B( upward arrow), D( upward arrow), C( downward arrow) as well as five alpha-helices I-V. The structures of most members of family PF02130 can be reliably constructed using the TM1509 NMR structure, demonstrating high leverage for exploration of fold space. A multiple sequence alignment of TM1509 with homologues of family UPF0054 shows that three polypeptide segments, as well as a putative zinc-binding consensus motif HGXLHLXGYDH located at the C-terminal end of alpha-helix IV, are highly conserved. The spatial arrangement of the three His residues of this UPF0054 consensus motif is similar to the arrangement found for the His residues in the HEXXHXXGXXH zinc-binding consensus motif of matrix metallo-proteases (MMPs). Moreover, the other conserved polypeptide segments form a large cavity which encloses the putative Zn-binding pocket and might confer specificity during catalysis. However, TM1509 and the other members of the UPF0054 family do not have the crucial Glu residue in position 2 of the MMP consensus motif. Intriguingly, the TM1509 structure indicates that the Asp in the UPF0054 consensus motif (Asp 111 in TM1509) may overtake the catalytic role of the Glu. This suggests that protein family UPF0054 might contain members of a hitherto uncharacterized class of metalloproteases.

Amino Acid Sequence↗
PubMed2005

Rapid NMR data collection.

Rapid data collection is an area of intense research in biomolecular NMR spectroscopy, in particular for high-throughput structure determination in structural genomics. NMR data acquisition and processing protocols for rapidly obtaining high-dimensional spectral information aim at avoiding sampling limited data collection and are reviewed here with emphasis on G-matrix Fourier transform NMR spectroscopy.

Data Collection↗
PubMed2005

An integrated platform for automated analysis of protein NMR structures.

Recent developments provide automated analysis of NMR assignments and three-dimensional (3D) structures of proteins. These approaches are generally applicable to proteins ranging from about 50 to 150 amino acids. In this chapter, we summarize progress by the Northeast Structural Genomics Consortium in standardizing the NMR data collection process for protein structure determination and in building an integrated platform for automated protein NMR structure analysis. Our integrated platform includes the following principal steps: (1) standardized NMR data collection, (2) standardized data processing (including spectral referencing and Fourier transformation), (3) automated peak picking and peak list editing, (4) automated analysis of resonance assignments, (5) automated analysis of NOESY data together with 3D structure determination, and (6) methods for protein structure validation. In particular, the software AutoStructure for automated NOESY data analysis is described in this chapter, together with a discussion of practical considerations for its use in high-throughput structure production efforts. The critical area of data quality assessment has evolved significantly over the past few years and involves evaluation of both intermediate and final peak lists, resonance assignments, and structural information derived from the NMR data. Methods for quality control of each of the major automated analysis steps in our platform are also discussed. Despite significant remaining challenges, when good quality data are available, automated analysis of protein NMR assignments and structures with this platform is both fast and reliable.

Data Interpretation, Statistical↗
PubMed2005

High-quality homology models derived from NMR and X-ray structures of E. coli proteins YgdK and Suf E suggest that all members of the YgdK/Suf E protein family are enhancers of cysteine desulfurases.

The structural biology of proteins mediating iron-sulfur (Fe-S) cluster assembly is central for understanding several important biological processes. Here we present the NMR structure of the 16-kDa protein YgdK from Escherichia coli, which shares 35% sequence identity with the E. coli protein SufE. The SufE X-ray crystal structure was solved in parallel with the YdgK NMR structure in the Northeast Structural Genomics (NESG) consortium. Both proteins are (1) key components for Fe-S metabolism, (2) exhibit the same distinct fold, and (3) belong to a family of at least 70 prokaryotic and eukaryotic sequence homologs. Accurate homology models were calculated for the YgdK/SufE family based on YgdK NMR and SufE crystal structure. Both structural templates contributed equally, exemplifying synergy of NMR and X-ray crystallography. SufE acts as an enhancer of the cysteine desulfurase activity of SufS by SufE-SufS complex formation. A homology model of CsdA, a desulfurase encoded in the same operon as YgdK, was modeled using the X-ray structure of SufS as a template. Protein surface and electrostatic complementarities strongly suggest that YgdK and CsdA likewise form a functional two-component desulfurase complex. Moreover, structural features of YgdK and SufS, which can be linked to their interaction with desulfurases, are conserved in all homology models. It thus appears very likely that all members of the YgdK/SufE family act as enhancers of Suf-S-like desulfurases. The present study exemplifies that "refined" selection of two (or more) targets enables high-quality homology modeling of large protein families.

Animals↗