Biomedical subjects

J H Prestegard

Publications and source records attributed to J H Prestegard.

At least 19 recordsLinked to original sources

PubMed2000

Nuclear magnetic resonance studies of the N-terminal fragment of adenosine diphosphate ribosylation factor 1 in micelles and bicelles: influence of N-myristoylation.

The N-terminal fragment of adenosine diphosphate (ADP) ribosylation factor 1 (ARF1) is proposed to be involved in the guanosine triphosphate- (GTP-) dependent, reversible association of the protein with membranes through the interaction of not only the N-linked myristoyl chain but also its highly conserved N-terminal hydrophobic residues. Based on the N-terminal sequence of this protein, specifically (13)C- and (15)N-labeled peptides were synthesized with and without an N-myristoyl anchor. The behavior, including structure, dynamics, and orientation, of these peptides in a lipid environment was then studied through a combination of solution (1)H nuclear magnetic resonance (NMR) techniques in micelles and heteronuclear solid-state NMR experiments in magnetically aligned bicelles. The work presented is an extension of the previously reported characterization of the myristoylated N-terminal fragment of ARF1 [Losonczi and Prestegard (1998) Biochemistry 37, 706-716] to include a comparison to a nonmyristoylated analogue. Results indicate that both myristoylated and nonmyristoylated peptides are alpha-helical in a lipid environment and that N-myristoylation does not greatly influence the structure of the peptides. Evidence is presented suggesting association of the peptides with bilayer disks through a combination of edge and surface interactions.

ADP-Ribosylation Factor 1↗

PubMed2000

Molecular symmetry as an aid to geometry determination in ligand protein complexes.

Dipole-dipole couplings between pairs of spin 12 nuclei, which can be measured from NMR spectra in field-ordered media, offer useful constraints on the orientation of various fragments in molecular systems. However, the orientation of fragments relative to a molecule fixed reference frame is often key to complete structure determination. Here, we demonstrate that the symmetry properties of molecular complexes can aid in the definition of a reference frame. It is shown that a threefold rotational symmetry axis dictates the direction and symmetry of the experimentally determined order tensor for alpha-methyl-mannose in fast exchange among the three symmetry-related binding sites of mannose binding protein. This approach facilitates studies of the geometry of the ligand in the protein-ligand complex and also may provide a novel route to structure determination of a homomultimeric protein.

Carrier Proteins↗

PubMed2000

Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings.

Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using high-resolution 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.

Bacterial Proteins↗

PubMed1999

Residual dipolar coupling derived orientational constraints on ligand geometry in a 53 kDa protein-ligand complex.

The geometric relationships between ligands and the functional groups that bind ligands in soluble ligand-protein complexes have traditionally been deduced from distance constraints between pairs of NMR active nuclei spanning the ligand-protein interface. Frequently, the steep inverse distance dependence of the nuclear Overhauser effect (NOE), from which the distance constraints are derived, makes identification of sufficient numbers of constraints difficult. In these cases the ability to supplement NOE-derived information with distance-independent angular information can be very important. Here, the observation of residual dipolar couplings from alpha-methyl mannose bound to mannose binding-protein in a dilute liquid crystalline medium has allowed the determination of a bound ligand's average orientation. The 3-fold rotational symmetry of mannose-binding protein defines its orientational tensor and obviates the need to determine experimentally the protein's average orientation. Through superimposition of ligand and protein orientational tensors we describe the binding geometry of alpha-methyl mannose bound to mannose-binding protein. This new method is of general applicability to the study of ligands bound to proteins, and it is of particular interest when neither X-ray crystallography nor NOE techniques can provide sufficient information to describe binding geometries.

Binding Sites↗

PubMed1999

Backbone dynamics of the N-terminal domain in E. coli DnaJ determined by 15N- and 13CO-relaxation measurements.

The backbone dynamics of the N-terminal domain of the chaperone protein Escherichia coli DnaJ have been investigated using steady-state 1H-15N NOEs, 15N T1, T2, and T1 rho relaxation times, steady-state 13C alpha-13CO NOEs, and 13CO T1 relaxation times. Two recombinant constructs of the N-terminal domain of DnaJ have been studied. One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ, and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition to the "J-domain". DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact that all currently identified sites responsible for DnaJ-DnaK interaction are located in this region. DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ. Recently, a structural analysis of these two molecules was presented in an effort to elucidate the origin of their functional differences [Huang, K., Flanagan, J. M., and Prestegard, J. H. (1999) Protein Science 8, 203-214]. Herein, an analysis of dynamic properties is presented in a similar effort. A generalized model-free approach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis of measured relaxation parameters. Our results show that internal motions on pico- to nanosecond time scales in the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformational exchange on micro- to millisecond time scales increases. We speculate that the enhanced flexibility of residues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPase stimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.

Anisotropy↗

PubMed1999

Domain orientation and dynamics in multidomain proteins from residual dipolar couplings.

The data most commonly available for the determination of macromolecular structures in solution are NOE based distance estimates and spin-spin coupling constant based dihedral angle estimates. This information is, unfortunately, inherently short-range in nature. Thus, for many multidomain proteins, little information is available to accurately position weakly interacting domains with respect to each other. Recent studies of proteins aligned in dilute liquid crystalline solvents have shown the utility of measuring anisotropic spin interactions, such as residual dipolar couplings, to obtain unique long-range structural information. In this work, the latter approach is taken to explore the relative domain orientation in a two-domain fragment from the protein barley lectin. An approach based on singular value decomposition as opposed to simulated annealing is used to directly determine order tensors for each domain from residual (15)N-(1)H dipolar couplings, and the limitations of the two approaches are discussed. Comparison of the order tensor principal axis frames as separately determined for each domain indicates that the two domains are not oriented as in the crystal structure of wheat germ agglutinin, a highly homologous protein ( approximately 95% sequence identical). Furthermore, differences in the order tensor values suggest that the two domains are not statically positioned but are experiencing different reorientational dynamics and, to a large degree, may be considered to reorient independently. Data are also presented that suggest that a specific association occurs between one domain and the lipid bicelles comprising the liquid crystal solvent.

Computer Simulation↗

PubMed1999

Order matrix analysis of residual dipolar couplings using singular value decomposition.

The measurement of anisotropic spin interactions, such as residual dipolar couplings, in partially ordered solutions can provide valuable information on biomolecular structure. While the information can be used to refine local structure, it can make a unique contribution in determining the relative orientation of remote parts of molecules, which are locally well structured, but poorly connected based on NOE data. Analysis of dipolar couplings in terms of Saupe order matrices provides a concise description of both orientation and motional properties of locally structured fragments in these cases. This paper demonstrates that by using singular value decomposition as a method for calculating the order matrices, principal frames and order parameters can be determined efficiently, even when a very limited set of experimental data is available. Analysis of 1H-15N dipolar couplings, measured in a two-domain fragment of the barley lectin protein, is used to illustrate the computational method.

Anisotropy↗

PubMed1999

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS).

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by 31P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7 degrees, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0 degree toward 54.7 degrees. Thus the relative sign of dipolar and scalar couplings can be determined.

Anisotropy↗

PubMed1999

The influence of C-terminal extension on the structure of the "J-domain" in E. coli DnaJ.

Two different recombinant constructs of the N-terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen-15 and carbon-13. One, DnaJ(1-78), contains the complete "J-domain," and the other, DnaJ(1-104), contains both the "J-domain" and a conserved "G/F" extension at the C-terminus. The three-dimensional structures of these proteins have been determined by heteronuclear NMR experiments. In both proteins the "J-domain" adopts a compact structure consisting of a helix-turn-helix-loop-helix-turn-helix motif. In contrast, the "G/F" region in DnaJ(1-104) does not fold into a well-defined structure. Nevertheless, the "G/F" region has been found to have an effect on the packing of the helices in the "J-domain" in DnaJ(1-104). Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures. In addition, there are some local conformational changes in the loop region connecting the two central helices. These structural differences in the "J-domain" in the presence of the "G/F" region may be related to the observation that DnaJ (1-78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1-78 segment.

Amino Acid Sequence↗

PubMed1998

Nuclear magnetic resonance characterization of the myristoylated, N-terminal fragment of ADP-ribosylation factor 1 in a magnetically oriented membrane array.

The behavior of the N-terminal fragment of human ADP-ribosylation factor 1 (ARF1) in a membranelike environment is described. This is accomplished using heteronuclear liquid crystal NMR techniques in a magnetically oriented membrane array on a selectively 13C- and 15N-labeled peptide. After full assignment of the labeled sites, residual dipolar couplings (13C-13C, 15N-1H and, 13C-15N) and chemical shift anisotropy effects (amide 13C and 15N) were measured. The experimental data were interpreted using order matrix calculations to yield orientational and dynamic information for four separate, rigid amide planes. The experimental data obtained proves that the amphipathic peptide interacts with the bilayer in a mode that is consistent with an alpha-helix having its axis parallel to the membrane surface. Possibilities of extending the employed techniques to larger and uniformly labeled systems are discussed.

ADP-Ribosylation Factor 1↗

PubMed1998

Nuclear magnetic resonance structural and ligand binding studies of BLBC, a two-domain fragment of barley lectin.

Plant lectins are useful targets for biophysical studies of protein-carbohydrate recognition, a process of general interest because of its many roles in human physiology. Here, nuclear magnetic resonance (NMR) based structural and carbohydrate binding data on a two-domain fragment of the normally four-domain barley lectin protein are presented. The structural data, while preliminary, clearly shows that the recombinantly produced simplified model system, called BLBC, retains a nativelike fold. However, unlike the full-length parent protein, which is dimeric, BLBC is shown by pulsed-field gradient NMR diffusion studies to be largely monomeric. Still, the fragment retains nativelike carbohydrate binding properties. These properties are examined in some detail using heteronuclear single quantum coherence (HSQC) NMR spectroscopy on a uniformly 15N-labeled sample. Ligand-induced chemical shift changes in the 1H-15N HSQC spectrum are monitored as 15N-labeled BLBC is titrated with increasing concentrations of the unlabeled carbohydrate, N,N',N"-triacetylchitotriose. Well-resolved resonances from the individual domains show that BLBC binds ligand at two distinct and independent ligand binding sites, one in each domain. Binding constants of (1.1 +/- 0.2) x 10(3) M-1 and (0.6 +/- 0.2) x 10(3) M-1 are determined for the B and C domain sites, respectively. These results are discussed in relation to ligand binding studies that have previously been carried out on a highly homologous protein, wheat germ agglutinin.

Amino Acid Sequence↗

PubMed1998

A Metropolis Monte Carlo implementation of bayesian time-domain parameter estimation: application to coupling constant estimation from antiphase multiplets.

The Bayesian perspective on statistics asserts that it makes sense to speak of a probability of an unknown parameter having a particular value. Given a model for an observed, noise-corrupted signal, we may use Bayesian methods to estimate not only the most probable value for each parameter but also their distributions. We present an implementation of the Bayesian parameter estimation formalism developed by G. L. Bretthorst (1990, J. Magn. Reson. 88, 533) using the Metropolis Monte Carlo sampling algorithm to perform the parameter and error estimation. This allows us to make very few assumptions about the shape of the posterior distribution, and allows the easy introduction of prior knowledge about constraints among the model parameters. We present evidence that the error estimates obtained in this manner are realistic, and that the Monte Carlo approach can be used to accurately estimate coupling constants from antiphase doublets in synthetic and experimental data.

Algorithms↗

PubMed1998

Measurement of cross correlation between dipolar coupling and chemical shift anisotropy in the spin relaxation of 13C, 15N-labeled proteins.

We present a simple method for extracting interference effects between chemical shift anisotropy (CSA) and dipolar coupling from spin relaxation measurements in macromolecules, and we apply this method to extracting cross-correlation rates involving interference of amide 15N CSA and 15N-1H dipolar coupling and interference of carbonyl 13C' CSA and 15N-13C' dipolar coupling, in a small protein. A theoretical basis for the interpretation of these rates is presented. While it proves difficult to quantitatively separate the structural and dynamic contributions to these cross-correlation rates in the presence of anisotropic overall tumbling and a nonaxially symmetric chemical shift tensor, some useful qualitative correlations of data with protein structure can be seen when simplifying assumptions are made.

Adenosine Triphosphatases↗

PubMed1998

Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross-relaxation.

We present heteronuclear two-dimensional methods for the analysis of the geometry of exchangeable protons on a protein-bound carbohydrate. By using a water-selective NOESY-HSQC, we observed cross-relaxation between carbohydrate hydroxyl protons and non-exchangeable ring protons in the complex of [13C6]-alpha-methyl-D-mannopyranoside with recombinant rat mannose binding protein. Using a simple kinetic model, we were able to explain the differences in the initial slopes of the resulting cross-relaxation buildup curves in terms of the geometry of the hydroxyl protons in the bound state. The hydroxyl rotamers consistent with our cross-relaxation data fit very well with predictions based on the crystal structure of MBP bound to a mannose-rich oligosaccharide. These methods should be applicable to other systems where both ligand exchange and water exchange are fast relative to the rate of cross-relaxation.

Animals↗

PubMed1998

Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules.

Dissolving biological macromolecules in dilute bicelle solutions, which form oriented liquid crystals in the presence of a magnetic field, permits measurement of anisotropic spin interactions such as dipolar couplings [Tjandra, N. and Bax, A., Science, 278, 1111-1114]. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. This paper demonstrates that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems. An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids.

Colloids↗

PubMed1998

New techniques in structural NMR--anisotropic interactions.

Structure determination of biomolecules by NMR has traditionally been based on nuclear Overhauser effects (NOEs). Now there are additional sources of information that can complement NOEs in cases where positioning of remote parts of molecules is important, and where extension to larger and more complex systems is desired.

Anisotropy↗

PubMed1998

Improved estimation of CSA-dipolar coupling cross-correlation rates from laboratory-frame relaxation experiments.

We have investigated the underlying assumptions in estimating cross-correlation rates between chemical shift anisotropy (CSA) and dipolar coupling mechanisms in a scalar-coupled two-spin IS system, from laboratory frame relaxation experiments. It has ben shown that for an arbitrary relaxation delay, the difference in relaxation rates of the individual components of an in-phase (or antiphase) doublet is not related to the CSA-dipolar coupling cross-correlation rate in a simple way. This is especially true in the case where the difference in the decay rates of the in-phase and antiphase terms of the density matrix becomes comparable to the magnitude of the scalar coupling between the two spins. Improved means of extracting cross-correlation rates in these cases are presented.

Anisotropy↗

PubMed1997

A transmembrane helix dimer: structure and implications.

The three-dimensional structure of the dimeric transmembrane domain of glycophorin A (GpA) was determined by solution nuclear magnetic resonance spectroscopy of a 40-residue peptide solubilized in aqueous detergent micelles. The GpA membrane-spanning alpha helices cross at an angle of -40 degrees and form a small but well-packed interface that lacks intermonomer hydrogen bonds. The structure provides an explanation for the previously characterized sequence dependence of GpA dimerization and demonstrates that van der Waals interactions alone can mediate stable and specific associations between transmembrane helices.

Amino Acid Sequence↗