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A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of diseases of the brain. Our previous studies demonstrated that that a soluble form of melanotransferrin (MTf; Uniprot P08582; also known as p97, MFI2, and CD228), a mammalian iron-transport protein, is an effective carrier for delivery of drug conjugates across the BBB into the brain and was the first BBB targeting delivery system to demonstrate therapeutic efficacy within the brain. Here, we performed a screen to identify peptides from MTf capable of traversing the BBB. We identified a highly conserved 12-amino acid peptide, termed MTfp, that retains the ability to cross the intact BBB intact, distributes throughout the parenchyma, and enter endosomes and lysosomes within neurons, astrocytes and microglia in the brain. This peptide may provide a platform for the transport of therapeutics to the CNS, and thereby offers new avenues for potential treatments of neuropathologies that are currently refractory to existing therapies. [ABSTRACT FROM AUTHOR]

T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H–T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl‐enzyme intermediate (Kuroki et al., PNAS 1999; 96:8949–8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H–T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H–T4L, including the active site Arg145, are positively charged under neutral pH conditions. Brief statement: The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pKa values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild‐type and mutant form of T4 lysozyme cleave bacterial peptidoglycan. [ABSTRACT FROM AUTHOR]

The pathogenic bacterium Salmonella enterica serovar Typhimurium utilizes two type III secretion systems (T3SS) to inject effector proteins into target cells upon infection. The T3SS secretion apparatus (the injectisome) is a large macromolecular assembly composed of over twenty proteins, many in highly oligomeric states. A sub‐structure of the injectisome, termed the basal body, spans both membranes and the periplasmic space of the bacterium. It is primarily composed of three integral membranes proteins, InvG, PrgH, and PrgK, that form ring structures through which components are secreted. In particular, PrgK possesses a periplasmic region consisting of two globular domains joined by a linker polypeptide. We showed previously that in isolation, this region adopts two distinct conformations, of with only one is observed in the assembled basal body complex. Here, using NMR spectroscopy, we further characterize these two conformations. In particular, we demonstrate that the interaction of the linker region with the first globular domain, as found in the intact basal body, is dependent upon the cis conformation of the Leu77‐Pro78 peptide. Furthermore, this interaction is pH‐dependent due to coupling with hydrogen bond formation between Tyr75 and His42 in its neutral Nδ1H tautomeric form. This pH‐dependent interaction may play a role in the regulation of the secretion apparatus disassembly in the context of bacterial infection. [ABSTRACT FROM AUTHOR]

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (p Ka value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic p Ka value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high p Ka value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group. [ABSTRACT FROM AUTHOR]

The p K values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the H, C, and N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein p K measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues. [ABSTRACT FROM AUTHOR]

The E26 transformation-specific (Ets-1) transcription factor is auto-inhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (Φ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-Φ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression. [ABSTRACT FROM AUTHOR]

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond H-N or H-C couplings. However, by filtering against protons directly bonded to C or N nuclei, signals from slowly-exchanging hydroxyls can be observed in the H-NMR spectrum of a uniformly C/N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with C/N-serine or C/N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via J couplings in long-range C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly C/N-labeled BcX using a quantitative C/N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a C/N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein. [ABSTRACT FROM AUTHOR]

Herein are reported the mainchain H, C and N chemical shift assignments and amide N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein. [ABSTRACT FROM AUTHOR]

Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, 15N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site. [ABSTRACT FROM AUTHOR]

We have developed NMR spectroscopic methods to investigate the tyrosines within Bacillus circulans xylanase (BcX). Four slowly exchanging buried tyrosine hydroxyl protons with chemical shifts between 7.5 and 12.5 ppm were found using a long-range C-HSQC experiment that exploits the J coupling between the ring H and C nuclei. The NMR signals from these protons were assigned via C-tyrosine selective labelling and a suite of scalar and C,N-filtered/edited NOE correlation spectra. Of the fifteen tyrosines in BcX, only the buried Tyr79 and Tyr105 showed four distinct, rather than two averaged, signals from ring C-H pairs, indicative of slow flipping on the chemical shift timescale. Ring flipping rate constants of ~10 and ~0.2 s were measured for the two residues, respectively, using a C longitudinal exchange experiment. The hydrogen bonding properties of the Tyr79 and Tyr105 hydroxyls were also defined by complementary NOE and J-coupling measurements. The H hydrogen-deuterium exchange rate constants of the buried tyrosines were determined from C/N-filtered spectra recorded as a function of pH. These exchange rate constants correspond to estimated protection factors of ~10-10 relative to a random coil tyrosine. The phenolic sidechain p K values were also measured by monitoring their pH-dependent C chemical shifts via H(C)C correlation spectra. Exposed tyrosines had unperturbed p K values of ~10.2, whereas buried residues remained predominantly neutral at or even above pH 11. Combined with selective isotope labelling, these NMR experiments should prove useful for investigating the structural and electrostatic properties of tyrosines in many interesting proteins. [ABSTRACT FROM AUTHOR]

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (p K values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain C nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic p K values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic p K values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined p K values and hence catalytic roles of these two residues result from their electrostatic coupling. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumorassociated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen." Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by. sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen. [ABSTRACT FROM AUTHOR]

A simple NMR method is presented for the identification and assignment of phosphorylated serine and threonine residues in 13C- or 13C/15N-labeled proteins. By exploiting modest (~5 Hz) 2- and 3-bond 13C–31P scalar couplings, the aliphatic 1H–13C signals from phosphoserines and phosphothreonines can be detected selectively in a 31P spin-echo difference constant time 1H-13C HSQC spectrum. Inclusion of the same 31P spin-echo element within the 13C frequency editing period of an intraHNCA or HN(CO)CA experiment allows identification of the amide 1HN and 15N signals of residues (i) for which 13Cα(i) or 13Cα(i − 1), respectively, are coupled to a phosphate. Furthermore, 31P resonance assignments can be obtained by applying selective low power cw 31P decoupling during the spin-echo period. The approach is demonstrated using a PNT domain containing fragment of the transcription factor Ets-1, phosphorylated in vitro at Thr38 and Ser41 with the MAP kinase ERK2. [ABSTRACT FROM AUTHOR]

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. ; Jung et al. ; Rotondi and Gierasch ), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca2+ or Mg2+, has not been fully characterized. It has, however, been suggested that addition of Ca2+ to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. ; Kopp et al. ), may also be important for Ca2+ binding (Jung et al. ). In this work, we have tried: (1) to further pinpoint how Ca2+ affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional 13C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca2+ and daptomycin. The 13C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg2+ are also presented. The implication of these results for the biological function of daptomycin is discussed. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca2+)-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca2+-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca2+-free CaM2/3 is predominantly unfolded, but upon binding Ca2+, adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel β-sheet. Despite having an 'even-odd' pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the 'odd-even' paired N- and C-domains of Ca2+-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca2+ binding with a Kd1 = 30 ± 5 μM to EF-3, and a Kd2 > 1000 μM to EF-2. Binding of the first equivalent of Ca2+ induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its 'even-odd' paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca2+ affinity exhibited by EF-2 in the context of CaM2/3. [ABSTRACT FROM AUTHOR]

The apolipoprotein A-I (apoA-I) solution structure in the presence of sodium dodecyl sulfate (SDS) was determined by combination of chemical shift index and torsion angle likelihood obtained from shift and sequence similarity methods. ApoA-I in lipid-mimetic solution is composed of α-helices (residues 8–32, 45–64, 67–77, 82–86, 90–97, 100–118, 122–140, 146–162, 167–205, 210–216 and 221–239), with 2–5 residue irregular segments between helical repeats, and the irregular segment 78–81 within helical repeat 2. ApoA-I is a monomer in the SDS complex and no evidence of interhelical interactions is found. Comparison of the apoA-I and apoA-I(1–186) [Okon et al., FEBS Lett. 487 (2001) 390–396] solution structures revealed that apoA-I undergoes a conformational change around Pro121. [Copyright &y& Elsevier]

Presents a letter to the editor about the role of the LexA repressor.