NMR structures of paramagnetic metalloproteins

Arnesano, Fabio; Banci, Lucia; Piccioli, Mario

doi:10.1017/s0033583506004161

Cited by 90 publications

(86 citation statements)

References 193 publications

(299 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 2 paramagnetic states can be distinguished from the g factors in the EPR spectra, which can be detected only at near-liquid helium temperature because of fast relaxation at ambient temperature (50). However, in NMR spectroscopy, the paramagnetic effect from [2Fe-2S] 2ϩ and [4Fe-4S] 2ϩ clusters causes a hyperfine shift in the NMR resonances, which can be detected (32,(51)(52)(53). A summary of the EPR results in the literature, combined with our results of the holo-KpFeoC, is presented in Table S1 in the supplemental material for comparison.…”

Section: Discussionmentioning

confidence: 99%

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Hsueh

Chen

et al. 2013

J Bacteriol

View full text Add to dashboard Cite

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe 2؉ ) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen-or iron-sensitive coordination of the Fe-S cluster.

show abstract

Section: Discussionmentioning

confidence: 99%

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Hsueh

Chen

et al. 2013

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form.…”

mentioning

confidence: 99%

“…Paramagnetic effects have long been used and today play a central role in solution-state NMR structural determinations in metalloproteins (14)(15)(16), or in biomolecules covalently modified with a spin-label (17)(18)(19). Importantly, paramagnetic effects can manifest themselves as either shifts (contact or "pseudocontact" shifts) or enhanced relaxation (PREs), depending on the character of the metal center.…”

mentioning

confidence: 99%

“…1 103 resonances were resolved in the CP-HSQC, indicating that the CP-HSQC experiment is an efficient detection block for 15 N or 13 C relaxation measurements. Thus, the 15 N-1 H CP-HSQC dipolar correlation module was combined with a 15 N inversion-recovery block (30,31), 15 N spin-lock (32,33), or additional 13 C-15 N specific transfers (34) and 13 C inversion-recovery (35) into a new set of extremely sensitive and resolved experiments. Pulse schemes are shown in Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Knight

Pell

Bertini

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

179

244

View full text Add to dashboard Cite

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1 H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1 H-1 H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.paramagnetism | nuclear relaxation rates | copper | microcrystal S tructure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6-8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9-11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined wit...

show abstract

“…13 C-13 C COSY crosspeaks are detectable only if their line width is smaller than ϳ300 Hz (48). A coordination bond induces a strong electron spin delocalization from the metal to the ligand affecting both chemical shift and nuclear relaxation (49), thus making the signals of the ligands undetectable in the 13 C-13 C COSY. This means that Tyr 75 is not a heme iron ligand in any of the two forms detected by NMR as a function of pH.…”

mentioning

confidence: 99%