Purple Membrane Induced Alignment of Biological Macromolecules in the Magnetic Field

Sass, J.K.; Cordier, Florence; Hoffmann, Adrian; Rogowski, Marco; Cousin, Anne; Omichinski, James G.; Löwen, and H.; Grzesiek, Stephan

doi:10.1021/ja983887w

Cited by 225 publications

(210 citation statements)

References 47 publications

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“…These changes in order tensor properties have previously been reported using different aligning reagents (17), changing pH in the presence of His-Tag on the protein (10), and by doping bicelle media with charged lipids (10,18). We use the latter approach for modulating order tensor properties.…”

Section: Introductionsupporting

confidence: 62%

Variation of Molecular Alignment as a Means of Resolving Orientational Ambiguities in Protein Structures from Dipolar Couplings

Al‐Hashimi

Valafar

Terrell

et al. 2000

Journal of Magnetic Resonance

165

174

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Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using highresolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on 15 N-1 H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media. © 2000 Academic Press Key Words: NMR; order matrix; bicelle; rubredoxin; protein fold. INTRODUCTIONThe measurement of residual dipolar couplings between protein backbone nuclei can provide an alternative to NOE information in the determination of protein folds (1)(2)(3)(4)(5)(6)(7)(8). These measurements can constrain the relative orientation of molecular fragments regardless of their separation in space, a fact of considerable value when studying loosely connected protein domains or backbone segments of proteins that are separated by unassigned side chain atoms (5,6,9). However, relative fragment orientations cannot be uniquely determined from a single set of residual dipolar coupling measurements, no matter how numerous the measurements (6, 9 -11). This limitation ultimately arises from the multi-valued character of angular dependent residual dipolar coupling function,where is the angle between the internuclear vector and the magnetic field (1, 12). For a single residual dipolar coupling measurement, it is easy to see that one can only constrain the orientation of an internuclear vector into two "cones" of orientation (10). More measurements restrict solutions to intersection of cones but a single allowed solution is never found (10).Using an order matrix analysis of multiple measurements from noncollinear vectors within a single fragment, it is easy to show that uncertainty in orientation reduces only to a fourfold degeneracy (6,9,(11)(12)(13)(14). In this analysis, residual dipolar coupling measurements are used to determine the five independent elements of a symmetric and traceless 3 ϫ 3 order matrix (11,13). These five parameters can be reformulated in terms of an alignment of axes (three angles) for a principal order frame, and values of a principal order parameter (S zz ), and an asymmetry parameter ( ϭ ͉(S xx Ϫ S yy )/S zz )͉) (11,13). Because the order parameters are themselves insensitive to axis inversion, there are four possible ways to direct a...

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Section: Introductionsupporting

confidence: 62%

Variation of Molecular Alignment as a Means of Resolving Orientational Ambiguities in Protein Structures from Dipolar Couplings

Al‐Hashimi

Valafar

Terrell

et al. 2000

Journal of Magnetic Resonance

165

174

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“…NMR samples were 0.5 mM for a fully 13 C, 15 N-labeled RNA strand and 2 mM for an unlabeled RNA strand in 0.01 mM EDTA͞10 mM sodium phosphate (pH 6.5). Purple membrane (PM, Munich Innovative Biomaterials, Marburg, Germany) was added to 2 mg͞ml for the measurement of residual dipolar couplings (11,12). NMR Spectroscopy.…”

Section: Rna Sample Preparationmentioning

confidence: 99%

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Bae

Cheong

Lee

et al. 2001

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

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The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5 and 3 ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A)⅐U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 ؎ 10°) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.T he influenza A virus, a member of the Orthomyxoviridae family, has a genome consisting of eight single-stranded RNA molecules of negative polarity. These eight RNA segments encode ten proteins, including three RNA-dependent RNA polymerase (RdRp) proteins (PA, PB1, PB2) and one nucleoprotein (NP). The transcription and replication of the viral genome are performed in the nucleus of infected cells by a ribonucleoprotein (RNP) complex that is composed of the three polymerase proteins, NP, and the viral RNA (vRNA). Replication of the vRNA produces a full-length copy of positive-sense RNA (cRNA). The cRNA is then used in the formation of another RNP complex, which serves to generate newly synthesized vRNA. Transcription is also performed by the same RNP complex; however, initiation occurs by the pirating of a 7-methyl guanosine cap structure from a host mRNA. Viral transcription produces an mRNA molecule that is 15 to 22 nucleotides (nt) shorter than cRNA and contains a poly(A) tail (1).The influenza A virus RdRp binds specifically to the partial duplex promoter (also referred to as the panhandle RNA), which is made from the 5Ј and 3Ј termini of each RNA genome segment. Within this partial duplex, 13 nt at the 5Ј end and 12 nt at the 3Ј end are highly conserved among most influenza A virus variants (Fig. 1A). All of the necessary signals for replication and genome packaging appear to reside in these terminal sequences (2), and several pieces of evidence imply a regulatory role for the terminal structure in viral transcription initiation (3-6), termination, and polyadenylation (7,8).The RdRps are for the most part virus-encoded polymerases that synthesize RNA from an RNA template without a DNA intermediate. Despite the unique features of RdRps, little has been deciphered about their mechanisms of promoter recognition and RNA synthesis. The promoter of the...

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“…19 Aside from liquid crystalline media, which mainly induce molecular alignment through collisional interactions, purple membrane or filamentous phage suspensions are also used to align the solute, primarily by weak electrostatic interactions. [28][29][30][31] Since both purple membrane and filamentous phage possess strong anisotropic magnetic susceptibility, they spontaneously align in a strong magnetic field (Figs. 1b and 1c).…”

Section: Chemical Shift Offset Induced By Weak Alignment Of the Moleculementioning

confidence: 99%

Anisotropic Nuclear Spin Interactions for the Morphology Analysis of Proteins in Solution by NMR Spectroscopy

Tate

2008

ANAL. SCI.

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NMR spectroscopy has become a standard method to determine the high-resolution structure of biomolecules, including protein, nucleic acids and their complexes.Conventional NMR structural determination is based on distance restraints obtained from proton-proton nuclear Overhauser effects (NOEs), which give approximate distances between interacting protons close together in space (< 5 -6 Å), and the torsion angles through vicinal spin couplings. 1 These NMR parameters all give shortrange structural information.Although the observable maximum distance by NOEs is limited, basically less than 5 Å, the short-range structural constraints can connect parts of a molecule that are far apart in the primary sequence, but close together in the three-dimensional coordinate of a globular protein. In this way, the short-range structural constraints successfully allowed accurate determination of secondary and tertiary molecular geometry of globular proteins.1 In spite of the great success of conventional NMR for the structural determination of globular proteins, there are significant limitations in determining multidomain protein structures that possess a hinge region situated between domains. In such cases, the number of proton distances may be insufficient to define the spatial arrangement of the respective domains. Furthermore, the NOE-derived distance restraints have limited accuracy; the parts defined by the sparse NOE interactions are not fully reliable. Thus, the relative positions of distant parts of extended or modular proteins are often poorly defined.In many multidomain proteins, the relative orientation of domains change in response to ligand binding or covalent modification, which includes phosphorylation, acetylation, methylation and so forth. Agency,[3][4][5] Chiyoda, Japan Determining the relative orientation of domains within a protein is an important problem in structural biology, which has been difficult to address by either X-ray crystallography or NMR. The structure of a multidomain protein in a crystal lattice can be altered by crystal packing forces, resulting in different domain arrangements from those in solution. On the other hand, conventional NMR primarily provides short-range structural information, including proton-proton distances derived from nuclear Overhauser effects (NOEs) and torsion angles through vicinal spin couplings. Thus, NMR cannot always determine the precise interdomain arrangements due to the sparsely observed spin interactions between domains. However, the weak alignment of proteins in solution has enabled a new NMR technique to determine the domain arrangement based on the different structural information, which defines the orientation of a structural unit in protein against the magnetic field. This technique relies on the anisotropic nuclear spin interactions that only occur for a molecule in a weakly aligned state. In this review, the basics of the new NMR approach are described with focusing on its application to domain orientation analysis. We also describe our recently est...

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Purple Membrane Induced Alignment of Biological Macromolecules in the Magnetic Field

Cited by 225 publications

References 47 publications

Variation of Molecular Alignment as a Means of Resolving Orientational Ambiguities in Protein Structures from Dipolar Couplings

Variation of Molecular Alignment as a Means of Resolving Orientational Ambiguities in Protein Structures from Dipolar Couplings

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Anisotropic Nuclear Spin Interactions for the Morphology Analysis of Proteins in Solution by NMR Spectroscopy

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