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N Tjandra

Publications and source records attributed to N Tjandra.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of slow interdomain motion of macromolecules using NMR relaxation data.

The interpretation of NMR relaxation data for macromolecules possessing slow interdomain motions is considered. It is shown how the "extended model-free approach" can be used to analyze (15)N backbone relaxation data acquired at three different field strengths for Xenopus Ca(2+)-ligated calmodulin. This protein is comprised of two domains connected by two rigid helices joined by a flexible segment. It is possible to uniquely determine all "extended model-free" parameters without any a priori assumptions regarding their magnitudes by simultaneously least-squares fitting the relaxation data measured at two different magnetic fields. It is found that the two connecting helices (and consequently the domains) undergo slow motions relative to the conformation in which the two helices are parallel. The time scales and amplitudes of these "wobbling" motions are characterized by effective correlation times and squared-order parameters of approximately 3 ns and 0.7, respectively. These values are consistent with independent estimates indicating that this procedure provides a useful first-order description of complex internal motions in macromolecules despite neglecting the coupling of overall and interdomain motions.

Animals↗
PubMed2001

Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein.

The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.

Amino Acid Sequence↗
PubMed2001

Simple multidimensional NMR experiments to obtain different types of one-bond dipolar couplings simultaneously.

In order to measure residual dipolar couplings, the molecule under study has to be partially oriented in the presence of the magnetic field. It has been observed that some protein samples are not stable under the conditions imposed by the orienting media. If different types of dipolar couplings are measured sequentially, their values will not agree with a unique alignment tensor that is changing slowly over time. This could bias the structure calculation. It would be more appropriate to obtain different types of dipolar couplings simultaneously, such that all the data correspond to one effective alignment tensor. We describe here a general NMR strategy designed to do so, that can be adapted to various existing pulse sequences.

Animals↗
PubMed2001

Measurement of 1H3'-31P dipolar couplings in a DNA oligonucleotide by constant-time NOESY difference spectroscopy.

The ratios of cross peak intensities in a selective constant-time NOESY experiment, recorded with and without 31P decoupling, yield values for the sum of the H3'-P scalar and dipolar couplings. The selective refocusing of H3' resonances in this experiment results in excellent resolution and sensitivity, even in the liquid crystalline phase where the 1H spectrum is broadened by unresolved homonuclear dipolar couplings. The vicinal H3'-P scalar and dipolar couplings in the DNA oligomer d(CGCGAATTCGCG)2 were measured in both isotropic solution, and in a liquid crystalline phase. Isotropic values are in good agreement with values reported previously. Dipolar couplings are in excellent agreement with the NMR structure for this dodecamer, and to a somewhat lesser extent with the X-ray structures.

Crystallography, X-Ray↗
PubMed2000

Structure of Bax: coregulation of dimer formation and intracellular localization.

Apoptosis is stimulated by the insertion of Bax from the cytosol into mitochondrial membranes. The solution structure of Bax, including the putative transmembrane domain at the C terminus, was determined in order to understand the regulation of its subcellular location. Bax consists of 9 alpha helices where the assembly of helices alpha1 through alpha 8 resembles that of the apoptosis inhibitor, Bcl-x(L). The C-terminal alpha 9 helix occupies the hydrophobic pocket proposed previously to mediate heterodimer formation and bioactivity of opposing members of the Bcl-2 family. The Bax structure shows that the orientation of helix alpha 9 provides simultaneous control over its mitochondrial targeting and dimer formation.

Amino Acid Sequence↗
PubMed2000

Chemical shift mapped DNA-binding sites and 15N relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K.

The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module-DNA interaction. In this study, the interaction of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K (KH3) with its cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, beta 1, and beta 2, are the primary regions involved in DNA binding for KH3. The nature of the KH3-ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton exchange experiments indicate that the two helices involved in DNA binding are less stable than other regions of secondary structure and that a large portion of KH3 backbone amide hydrogens are protected in some manner upon ssDNA binding. The major backbone dynamics features of KH3 are similar to those of the structurally comparable human papillomavirus-31 E2 DNA binding domain. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the protein fold.

Amides↗
PubMed2000

Direct refinement against proton-proton dipolar couplings in NMR structure determination of macromolecules.

The computational tools necessary for making use of (1)H-(1)H dipolar couplings in macromolecular structure refinement are presented. Potentials are described for direct refinement against (1)H-(1)H dipolar couplings of known sign as well as of unknown sign. In addition, a multiple potential is developed for prochiral protons whose stereospecific assignments are unknown. The utility of direct (1)H-(1)H dipolar coupling refinement is illustrated using the small protein ubiquitin. It is shown that direct (1)H-(1)H dipolar coupling refinement leads to improvements in the precision, accuracy, and quality of the resulting structures.

Macromolecular Substances↗
PubMed2000

Protein backbone (15)N relaxation rates as a tool for the diagnosis of structure quality.

In the work reported herein we define a structure validation factor that depends on protein backbone (15)N relaxation rates. This is an alternative method to the previously defined quality factors derived from anisotropic chemical shifts or residual dipolar couplings. We have used the structure dependence of (15)N relaxation rates of anisotropically tumbling proteins to calculate this structure diagnosis factor and have used it to demonstrate the improvement of protein structures refined with residual dipolar couplings.

Anisotropy↗
PubMed1999

Solution structure of human GAIP (Galpha interacting protein): a regulator of G protein signaling.

The solution structure of the human protein GAIP (Galpha interacting protein), a regulator of G protein signaling, has been determined by NMR techniques. Dipolar couplings of the oriented protein in two different liquid crystal media have been used in the structure calculation. The solution structure of GAIP is compared to the crystal structure of an homologous protein from rat (RGS4) complexed to the alpha-subunit of a G protein. Some of RGS4 residues involved in the Galpha-RGS binding interface have similar orientations in GAIP (free form), indicating that upon binding these residues do not suffer conformational rearrangements, and therefore, their role does not seem to be restricted to Galpha interaction but also to RGS folding and stability. We suggest that other structural differences between the two proteins may be related to the process of binding as well as to a distinct efficiency in their respective GTPase activating function.

Amino Acid Sequence↗
PubMed1999

High precision solution structure of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K, a c-myc transcription factor.

Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26-->Arg mutant of the C-terminal K-homology (KH) domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded beta-sheet stacked against three alpha-helices, two of which are nearly parallel to the strands of the beta-sheet. The Gly26-->Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the beta-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.

Amino Acid Sequence↗
PubMed1999

The use of dipolar couplings for determining the solution structure of rat apo-S100B(betabeta).

The relative orientations of adjacent structural elements without many well-defined NOE contacts between them are typically poorly defined in NMR structures. For apo-S100B(betabeta) and the structurally homologous protein calcyclin, the solution structures determined by conventional NMR exhibited considerable differences and made it impossible to draw unambiguous conclusions regarding the Ca2+-induced conformational change required for target protein binding. The structure of rat apo-S100B(betabeta) was recalculated using a large number of constraints derived from dipolar couplings that were measured in a dilute liquid crystalline phase. The dipolar couplings orient bond vectors relative to a single-axis system, and thereby remove much of the uncertainty in NOE-based structures. The structure of apo-S100B(betabeta) indicates a minimal change in the first, pseudo-EF-hand Ca2+ binding site, but a large reorientation of helix 3 in the second, classical EF-hand upon Ca2+ binding.

Animals↗
PubMed1998

Dynamical behavior of the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NC) contains two CCHC-type zinc knuckle domains that are essential for genome recognition, packaging and infectivity. The solution structure of the protein has been determined independently by three groups. Although the structures of the individual zinc knuckle domains are similar, two of the studies indicated that the knuckles behave as independently folded, non-interacting domains connected by a flexible tether, whereas one study revealed the presence of interknuckle NOE cross-peaks, which were interpreted in terms of a more compact structure in which the knuckles are in close proximity. We have collected multidimensional NMR data for the recombinant, isotopically labeled HIV-1 NC protein, and confirmed the presence of weak interknuckle NOEs. However, the NOE data are not consistent with a single protein conformation. 15N NMR relaxation studies reveal that the two zinc knuckle domains possess different effective rotational correlation times, indicating that the knuckles are not tumbling as a single globular domain. In addition, the 1H NMR chemical shifts of isolated zinc knuckle peptides are very similar to those of the intact protein. The combined results indicate that the interknuckle interactions, which involve the close approach of the side-chains of Phe16 and Trp37, are transitory. The solution behavior of NC may be best considered as a rapid equilibrium between conformations with weakly interacting and non-interacting knuckle domains. This inherent conformational flexibility may be functionally important, enabling adaptive binding of NC to different recognition elements within the HIV-1 psi-RNA packaging signal.

HIV-1↗
PubMed1998

Direct structure refinement against residual dipolar couplings in the presence of rhombicity of unknown magnitude.

Residual dipolar couplings arising from small degrees of alignment of molecules in a magnetic field provide unique long-range structural information. The potential of this approach for structure refinement has recently been demonstrated for a protein-DNA complex in which the magnetic susceptibility tensor was axially symmetric. For most macromolecules and macromolecular complexes, however, axial symmetry cannot be assumed. Moreover, the presence of significant rhombicity will clearly affect the accuracy of the resulting coordinates. In this Communication we present a simple calculational strategy that makes use of simulated annealing refinement against the residual dipolar couplings in combination with a grid search, to simultaneously refine the structures and ascertain the magnitude of the axial and rhombic components of the tensor.

Algorithms↗