Frits Abildgaard

1H, 13C and 15N chemical shift referencing in biomolecular NMR.

SummaryA considerable degree of variability exists in the way that 1H, 13C and 15N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common 1H, 13C and 15N chemical shift standards, now used in biomolecular NMR, to those proposed here.

Structure of outer membrane protein A transmembrane domain by NMR spectroscopy

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded β-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C8E4) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.

PRACTICAL INTRODUCTION TO THEORY AND IMPLEMENTATION OF MULTINUCLEAR, MULTIDIMENSIONAL NUCLEAR MAGNETIC RESONANCE EXPERIMENTS

Publisher Summary The objectives of this chapter are 2-fold. First, it presents basic unifying features of pulse sequences so that the underlying mechanics of even complicated sequences become more transparent. Second, a step-by-step guide to present the practical implementation and processing of multidimensional experiments is illustrated. Much progress has resulted from generalization of heteronuclear twodimensional (2D) NMR experiments with 13 C- and 15 N-labeled biomolecules to higher dimensions. The ultimate goal of NMR investigations of biomolecules is to obtain structural and dynamic information. To this end, many specialized experimental techniques similar to those described above have been developed, which allow the measurement of parameters that provide distance and dihedral angle constraints. In conjunction with the methods described in this chapter, computer-automated resonance assignments and spectral analysis techniques should facilitate efficient studies of larger biomolecules and are expected to accelerate the pace of NMR contributions to structural biochemistry.

The Ig fold of the core binding factor α Runt domain is a member of a family of structurally and functionally related Ig-fold DNA-binding domains

BACKGROUND CBFA is the DNA-binding subunit of the transcription factor complex called core binding factor, or CBF. Knockout of the Cbfa2 gene in mice leads to embryonic lethality and a profound block in hematopoietic development. Chromosomal disruptions of the human CBFA gene are associated with a large percentage of human leukemias. RESULTS Utilizing nuclear magnetic resonance spectroscopy we have determined the three-dimensional fold of the CBFA Runt domain in its DNA-bound state, showing that it is an s-type immunoglobulin (Ig) fold. DNA binding by the Runt domain is shown to be mediated by loop regions located at both ends of the Runt domain Ig fold. A putative site for CBFB binding has been identified; the spatial location of this site provides a rationale for the ability of CBFB to modulate the affinity of the Runt domain for DNA. CONCLUSIONS Structural comparisons demonstrate that the s-type Ig fold found in the Runt domain is conserved in the Ig folds found in the DNA-binding domains of NF-kappaB, NFAT, p53, STAT-1, and the T-domain. Thus, these proteins form a family of structurally and functionally related DNA-binding domains. Unlike the other members of this family, the Runt domain utilizes loops at both ends of the Ig fold for DNA recognition.

Structure, dynamics and function of the outer membrane protein A (OmpA) and influenza hemagglutinin fusion domain in detergent micelles by solution NMR

Recent progress from our laboratories to determine structures of small membrane proteins (up to 20 kDa) in detergent micelles by solution nuclear magnetic resonance (NMR) is reviewed. NMR opens a new window to also study, for the first time, the dynamics of membrane proteins. We report on recent attempts to correlate dynamic measurements on OmpA with the ion channel function of this protein. We also summarize how NMR and spin‐label electron paramagnetic resonance spectroscopy and selective mutagenesis can be combined to provide a structural basis towards understanding the mechanism of influenza hemagglutinin‐mediated membrane fusion.

Lipid Binding Ridge on Loops 2 and 3 of the C2A Domain of Synaptotagmin I as Revealed by NMR Spectroscopy

The C2A domain of synaptotagmin I, which binds Ca2+ and anionic phospholipids, serves as a Ca2+ sensor during excitation-secretion coupling. We have used multidimensional NMR to locate the region of C2A from rat synaptotagmin I that interacts, in the presence of Ca2+, with phosphatidylserine. Untagged, recombinant C2A was double-labeled with 13C and15N, and triple-resonance NMR data were collected from C2A samples containing either Ca2+ alone or Ca2+ plus 6:0 phosphatidylserine. Phospholipid binding led to changes in chemical shifts of backbone atoms in residues Arg233 and Phe234 of loop 3 (a loop that also binds Ca2+) and His198, Val205, and Phe206 of loop 2. These residues lie along a straight line on a surface ridge of the C2A domain. The only other residue that exhibited appreciable chemical shift changes upon adding lipid was His254; however, because His254 is located on the other side of the molecule from the phospholipid docking site defined by the other residues, its shifts may result from nonspecific interactions. The results show that the “docking ridge” responsible for Ca2+-dependent membrane association is localized on the opposite side of the C2A domain from the transmembrane and C2B domains of synaptotagmin.

1H, 13C and 15N NMR assignments and solution secondary structure of rat Apo-S100β

SummaryThe 1H, 13C and 15N NMR assignments of the backbone and side-chain resonances of rat S100β were made at pH 6.5 and 37°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE correlations, together with amide exchange rate and 1Hα, 13Cα and 13Cβ chemical shift data, provided extensive secondary structural information. Thus, the secondary structure of S100β was determined to comprise four helices (Leu3-Ser18, helix I; Lys29-Leu40, helix II; Gln50-Glu62, helix III; and Phe70-Ala83, helix IV), four loops (Gly19-His25, loop I; Ser41-Glu49, loop II; Asp63-Gly66, loop III; and Cys84-Glu91, loop IV) and two β-strands (Lys26-Lys28, β-strand I and Glu67-Asp69, β-strand II). The β-strands were found to align in an antiparallel manner to form a very small β-sheet. This secondary structure is consistent with predictions that S100β contains two ‘helix-loop-helix’ Ca2+-binding motifs known as EF-hands. The alignment of the β-sheet, which brings the two EF-hand domains of S100β into close proximity, is similar to that of several other Ca2+ ion-binding proteins.

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: nuclease-H124L and its ternary complex with Ca2+ and thymidine-3',5'-bisphosphate.

The solution structures of staphylococcal nuclease (nuclease) H124L and itsternary complex, (nuclease-H124L)•pdTp•Ca2+, were determinedby ab initio dynamic simulated annealing using 1925 NOE, 119 φ, 20χ1 and 112 hydrogen bond constraints for the free protein,and 2003 NOE, 118 φ, 20 χ1 and 114 hydrogen bondconstraints for the ternary complex. In both cases, the final structuresdisplay only small deviations from idealized covalent geometry. In structuredregions, the overall root-mean-square deviations from mean atomic coordinatesare 0.46 (±0.05) Å and 0.41 (±0.05) Å for thebackbone heavy atoms of nuclease and its ternary complex, respectively. Thebackbone conformations of residues in the loop formed byArg81–Gly86, which is adjacent to the activesite, are more precisely defined in the ternary complex than in unligatednuclease. Also, the protein side chains that show NOEs and evidence forhydrogen bonds to pdTp (Arg35, Lys84,Tyr85, Arg87, Tyr113, andTyr115) are better defined in the ternary complex. As has beenobserved previously in the X-ray structures of nuclease-WT, the binding ofpdTp causes the backbone of Tyr113 to change from an extendedto a left-handed α-helical conformation. The NMR structures reportedhere were compared with available X-ray structures: nuclease-H124L [Truckseset al. (1996) Protein Sci., 5, 1907–1916] and the ternary complex ofwild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct.Funct. Genet., 5, 183–201]. Overall, the solution structures ofnuclease-H124L are consistent with these crystal structures, but smalldifferences were observed between the structures in the solution and crystalenvironments. These included differences in the conformations of certain sidechains, a reduction in the extent of helix 1 in solution, and many fewerhydrogen bonds involving side chains in solution.

Applicability of magnetization transfer nuclear magnetic resonance to study chemical exchange reactions

Publisher Summary This chapter discusses the magnetization transfer technique in its general form. This includes cases where the relaxation rates of all exchanging signals are different, unintentional signal perturbations occur, and the exponents of the involved multi exponential functions are almost identical. Also cases with phase- and baseline- distorted spectra, low signal-to-noise ratios, and partly overlapping signals are considered. In particular, this chapter shall concentrate on the experimental procedure necessary to obtain spectra that are sufficiently informative, and the data analysis that must be used to retrieve the maximum information from these spectra. In nuclear magnetic resonance (NMR) studies of enzymes and other biological systems, overlapping signals often hamper the analysis. Because of the limited dynamic range of the spectrometer, further problems can arise when weak signals from biological molecules are present together with a strong solvent signal, in particular, the proton signal from water. Although effective procedures for solvent suppression are available, the application of these procedures may often result in phase- and baseline-distorted spectra preventing a correct evaluation of the signal intensities from the spectra.

NMR Studies of Retinoid-Protein Interactions: The Conformation of [13C]-β-Ionones Bound to β-Lactoglobulin B

AbstractPurpose. Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein β-lactoglobulin B (β-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. β-LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle. Methods. To examine the conformation of the model retinoid β-ionone both in solution and when bound to β-LG, NMR and computational methods have been employed. Results. Taken together, NMR studies of β-ionone in solution measuring scalar and dipolar coupling, as well as CHARMm calculations, suggest β-ionone prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR studies of l3C-labeled β-ionones bound to β-LG, primarily employing the HMQC-NOE experiment, suggest β-ionone also binds to β-LG in its 6-s-cis conformation. Conclusions. The methods employed here allow estimates of protein-bound ligand conformation. However, additional sites of ligand labeling will be necessary to aid in binding site localization.