Thomas Szyperski

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.

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.

Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools

Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity triple resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput triple resonance NMR for determining resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.Abbreviations: BPTI – bovine pancreatic trypsin inhibitor; LP – linear prediction; FT – Fourier transform; S/N – signal-to-noise ratio; FID – free induction decay

Protein dynamics in supercooled water: The search for slow motional modes

The impact of studying protein dynamics in supercooled water for identifying slow motional modes on the μs time scale is demonstrated. Backbone 15N spin relaxation parameters were measured at −13 °C for ubiquitin, which plays a central role for signaling proteolysis, cellular trafficking and kinase activation in eukaryotic organisms. A hitherto undetected motional mode involving Val 70 was found, which may well play an important role for ubiquitin recognition. The measurement of rotating frame 15N relaxation times as a function of the spin-lock field allowed determination of the correlation time of this motional mode, which would not have been feasible above 0 °C.

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 α+β fold comprising a four stranded β-sheet with topology A(↑), B(↑), D(↑), C(↓) as well as five α-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 α-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.

Simultaneously cycled NMR spectroscopy

Simultaneously cycled (SC) NMR was introduced and exemplified by implementing a set of 2-D [1H,1H] SC exclusive COSY (E.COSY) NMR experiments, that is, rf pulse flip-angle cycled (SFC), rf pulse phase cycled (SPC), and pulsed field gradient (PFG) strength cycled (SGC) E.COSY. Spatially selective 1H rf pulses were applied as composite pulses such that all steps of the respective cycles were affected simultaneously in different slices of the sample. This increased the data acquisition speed for an n-step cycle n-fold. A high intrinsic sensitivity was achieved by defining the cycles in a manner that the receiver phase remains constant for all steps of the cycle. Then, the signal resulting from applying the cycle corresponded to the sum of the signals from all steps of the cycle. Hence, the detected free induction decay did not have to be separated into the contributions arising from different slices, and read-out PFGs, which not only greatly reduce sensitivity but also negatively impact lineshapes in the direct dimension, were avoided. The current implementation of SFC E.COSY reached approximately 65% of the intrinsic sensitivity of the conventional phase cycled congener, making this experiment highly attractive whenever conventional data acquisition is sampling limited. Highly resolved SC E.COSY yielding accurate 3J-coupling values was recorded for the 416 Da plant alkaloid tomatidine within 80 min, that is, 12 times faster than with conventional phase cycled E.COSY. SC NMR is applicable for a large variety of NMR experiments and thus promises to be a valuable addition to the arsenal of approaches for tackling the NMR sampling problem to avoid sampling limited data acquisition.

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔCp 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 1H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, fu, 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°C and 39°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, ΔCp 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.

NMR structure of the 18 kDa protein CC1736 from Caulobacter crescentus identifies a member of the START domain superfamily and suggests residues mediating substrate specificity.

Yang Shen, Sharon Goldsmith-Fischman, Hanudatta S. Atreya, Thomas Acton, LiChung Ma, Rong Xiao, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, New York Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Northeast Structural Genomics Consortium

The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high‐quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX2CX14‐16CX2C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three‐dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a β‐sandwich consisting of two three‐stranded sheets with topology B(↓), A(↑), F(↓), and C(↑), D(↓), E(↑). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26‐residue amino‐terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy‐terminal core and the amino‐terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27.

NMR structure of the hypothetical protein AQ-1857 encoded by the Y157 gene from Aquifex aeolicus reveals a novel protein fold.

Duanxiang Xu, Gaohua Liu, Rong Xiao, Tom Acton, Sharon Goldsmith-Fischman, Barry Honig, Gaetano T. Montelione, and Thomas Szyperski* Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York Center of Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York Northeast Structural Genomics Consortium