Structural investigations connect the disordered N-terminal extension of HypB to the activities of HypB and SlyD in E. coli.

The activities of [NiFe]-hydrogenase enzymes, which are critical to many microbes, require insertion of a Ni(II) ion into the bimetallic catalytic center. Delivery of Ni(II) to [NiFe]-hydrogenases depends, in part, on the metallochaperone HypB, which lies at the center of a Ni(II) transfer pathway that includes the metal storage protein SlyD and the metallochaperone HypA. SlyD is a source of Ni(II) ions for HypB, whereas Ni(II) from HypB is transferred to HypA.

NMR Experiments for Studies of Dilute and Condensed Protein Phases: Application to the Phase-Separating Protein CAPRIN1.

Intrinsically disordered proteins (IDPs) or regions of intrinsic disorder in otherwise folded proteins (IDRs) play important roles in many different biological processes, including formation of biological condensates via liquid-liquid phase separation. NMR spectroscopy is a powerful tool for obtaining site-specific structural and dynamical information on IDPs/IDRs, and recent efforts have focused on the development of experiments for atomic-resolution studies of these molecules. These include triple-resonance experiments that are based on CO-direct detection of magnetization, exploiting increased sensitivity of cryogenically cooled probes.

Interplay of buried histidine protonation and protein stability in prion misfolding.

Misofolding of mammalian prion proteins (PrP) is believed to be the cause of a group of rare and fatal neurodegenerative diseases. Despite intense scrutiny however, the mechanism of the misfolding reaction remains unclear. We perform nuclear Magnetic Resonance and thermodynamic stability measurements on the C-terminal domains (residues 90-231) of two PrP variants exhibiting different pH-induced susceptibilities to aggregation: the susceptible hamster prion (GHaPrP) and its less susceptible rabbit homolog (RaPrP).

Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1.

The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K (K) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening.

Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch.

Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function.

Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions.

Intrinsically disordered proteins play crucial roles in regulatory processes and often function as protein interaction hubs. Here, we present a detailed characterization of a full-length disordered hub protein region involved in multiple dynamic complexes. We performed NMR, CD, and fluorescence binding studies on the nonphosphorylated and highly PKA-phosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) regulatory region, a ∼200-residue disordered segment involved in phosphorylation-dependent regulation of channel trafficking and gating.

Measurement of signs of chemical shift differences between ground and excited protein states: a comparison between H(S/M)QC and R1rho methods.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data.

Structural studies of FF domains of the transcription factor CA150 provide insights into the organization of FF domain tandem arrays.

FF domains are poorly understood protein interaction modules that are present within eukaryotic transcription factors, such as CA150 (TCERG-1). The CA150 FF domains have been shown to mediate interactions with the phosphorylated C-terminal domain of RNA polymerase II (phosphoCTD) and a multitude of transcription factors and RNA processing proteins, and may therefore have a central role in organizing transcription. FF domains occur in tandem arrays of up to six domains, although it is not known whether they adopt higher-order structures.

Measuring the signs of 1H(alpha) chemical shift differences between ground and excited protein states by off-resonance spin-lock R(1rho) NMR spectroscopy.

Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical shift differences between NMR probes in the exchanging states, |Delta omega|, are also extracted. Herein, we present a simple experiment for obtaining the sign of (1)H(alpha) Delta omega values by measuring off-resonance (1)H(alpha) decay rates, R(1rho), using weak proton spin-lock fields.

Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d.

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions.

Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy.

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains.

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.

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain.

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide.

Side-chain dynamics of the SAP SH2 domain correlate with a binding hot spot and a region with conformational plasticity.

X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to the Tyr.

Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g.

A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported.

A "three-pronged" binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome.

The SH2 domain protein SAP/SH2D1A, encoded by the X-linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation-independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified, T/S-x-x-x-x-V/I, where x represents any amino acid. Remarkably, this motif contains neither a Tyr nor a pTyr residue, a hallmark of conventional SH2 domain-ligand interactions.