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J H Prestegard

Publications and source records attributed to J H Prestegard.

At least 19 recordsLinked to original sources
PubMed2006

Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction.

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.

Archaeal Proteins↗
PubMed2006

Structural monitoring of oligosaccharides through 13C enrichment and NMR observation of acetyl groups.

Structural characterization of biomolecules by NMR methods frequently requires the enrichment of magnetically active isotopes at particular molecular sites. Introduction is usually achieved biosynthetically through the use of bacterial cultures grown on isotopically enriched media, but for certain types of molecules-cell-surface carbohydrates of mammalian origin, for example-this is not practical. Here we explore a means of introducing 13C-enriched sites, postisolation in natural carbohydrate products, and illustrate an ability to acquire sufficient information to select appropriate conformational models from among energetically allowed sets. The application presented involves replacement of native N-acetyl groups with 13C-labeled acetyl groups in a simple disaccharide derivative, (GlcNAc)2-OBu, or O-butyl-chitobiose. The assignment of the two acetyl groups introduced is based on a novel combination of NMR and mass spectrometry data. Structural information is obtained from chemical shift anisotropy offsets of 13C carbonyl resonances and 13C-13C dipolar couplings between the labeled methyl and carbonyl carbons of the acetyl groups. Although the application is to a relatively simple system, it lays the groundwork for application to biologically important complex carbohydrate systems.

Acetylation↗
PubMed2005

Determination of protein backbone structures from residual dipolar couplings.

There are a number of circumstances in which a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative in which computational modeling of many sequentially related structures from the backbone of a single family representative is anticipated. It is, however, also the case when the backbone may be a stepping-stone to more targeted studies of ligand interaction or protein-protein interaction. Here an NMR protocol is described that can produce a backbone structure of a protein without the need for extensive experiments directed at side chain resonance assignment or the collection of structural information on side chains. The procedure relies primarily on orientational constraints from residual dipolar couplings as opposed to distance constraints from NOEs. Procedures for sample preparation, data acquisition, and data analysis are described, along with examples from application to small target proteins of a structural genomics project.

Carbon Isotopes↗
PubMed2004

Backbone solution structures of proteins using residual dipolar couplings: application to a novel structural genomics target.

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods.

Amino Acid Sequence↗
PubMed2004

Quantitation of rapid proton-deuteron amide exchange using hadamard spectroscopy.

The rates of amide proton exchange in protein backbones are very useful reporters of accessibility and structural stability of specific residues and secondary structure elements. Measurement by monitoring changes in intensity of cross-peaks in standard (15)N-(1)H HSQC spectra as protons are replaced by solvent deuterons has become widely accepted. However, these methods are limited to relatively slow rates due to time limitations of the conventional 2D HSQC experiment. Here we show that a Hadamard encoded version of the HSQC, which relies on a multiplexed, frequency selective, excitation in the (15)N dimension, extends application to rates that are as much as an order of magnitude faster than those previously accessible.

Amides↗
PubMed2003

Conformational differences in liganded and unliganded states of Galectin-3.

The conformation of the carbohydrate recognition domain of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems, has been investigated in the absence of ligand and in the presence of N-acetylactosamine. A new methodology based on the measurement of residual dipolar couplings from NMR spectra has been used to characterize differences in protein structure along the backbone in the presence and absence of ligand, as well as the binding geometry of the ligand itself. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. The implications for ligand specificity and protein functionality are discussed.

Amino Sugars↗
PubMed2001

A dipolar coupling based strategy for simultaneous resonance assignment and structure determination of protein backbones.

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This approach relies on recent advances in high-resolution NMR spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned in field ordered media. Residual dipolar couplings are used for both geometric information and a filter in the assembly of residues in a sequential manner. Experimental data were collected in less than one week on a small redox protein, rubredoxin, that was 15N enriched but not enriched above 1% natural abundance in 13C. Given the acceleration possible with partial 13C enrichment, the protocol described should provide a very rapid route to protein structure determination. This is critical for the structural genomics initiative where protein expression and structural determination in a high-throughput manner will be needed.

Models, Molecular↗
PubMed2001

Nuclear magnetic resonance in the era of structural genomics.

Current interests in structural genomics, and the associated need for high through-put structure determination methods, offer an opportunity to examine new nuclear magnetic resonance (NMR) methodology and the impact this methodology can have on structure determination of proteins. The time required for structure determination by traditional NMR methods is currently long, but improved hardware, automation of analysis, and new sources of data such as residual dipolar couplings promise to change this. Greatly improved efficiency, coupled with an ability to characterize proteins that may not produce crystals suitable for investigation by X-ray diffraction, suggests that NMR will play an important role in structural genomics programs.

Animals↗
PubMed2001

Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum.

The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the origin of thermostability. In the native, metal (Fe or Zn) containing form, both proteins can maintain native structure at very high temperatures (>70 degrees C) for extended periods of time. Significant changes in either structure or backbone dynamics between 25 and 70 degrees C are not apparent for either protein. A kinetic difference with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermodynamic differences difficult to monitor. In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability. Below denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native holo form, except immediately adjacent to the metal binding site. In contrast, apo-CpRd is found to have a structure distinctly different from that of its holo form at low temperatures. This structure is rapidly lost upon heating, unfolding at approximately 40 degrees C. A PfRd mutant with the hydrophobic core mutated to match that of CpRd shows no change in thermostability in the metal-free state. A metal-free chimera with residues 1-15 of CpRd and the remaining 38 residues of PfRd is severely destabilized and is unfolded at 25 degrees C. Hence, the hydrophobic core does not seem to be the key determinant of thermostability; instead, data point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a possible contributor to the thermostability.

Amino Acid Sequence↗
PubMed2001

Structural and dynamic analysis of residual dipolar coupling data for proteins.

The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.

Humans↗
PubMed2001

Conformational analysis of a flexible oligosaccharide using residual dipolar couplings.

We present a new approach to the analysis of the conformational and the motional properties of an oligosaccharide, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside. The approach relies on an order matrix analysis of residual dipolar couplings in the solution state. By combining a number of different types of couplings, (1)D(CH), (2)D(CH), and D(HH), an order matrix is solved for each ring of the trimannoside. The resulting order parameters indicate the internal motion at the alpha (1,3) linkage to be limited, while significant motion is suggested at the alpha (1,6) linkage. Two structures for the trimannoside were determined by aligning the order tensor principal axes obtained from two different orienting media, bicelles and phage. The very similar conformations at the alpha (1,3) linkage of these two structures confirm that the internal motion at the alpha (1,3) linkage is small and the conformation is a good representation of a single preferred structure. The different conformations at the alpha (1,6) linkage suggest that the motional amplitudes are large and the conformations must be viewed as virtual conformers. Compared with traditional NMR methods, data acquisition is easy and data analysis is straightforward.

Carbohydrate Conformation↗
PubMed2001

Partial alignment of biomolecules: an aid to NMR characterization.

Partial alignment of biomolecules in solution has added a new dimension to structural investigation by high-resolution NMR methods. Applications to proteins, nucleic acids and carbohydrates now abound. Limitations initially associated with compatibility of biomolecules with the liquid-crystal media commonly used to achieve alignment have begun to disappear. This is, in part, a result of the introduction of a wide variety of new media. Future applications to biologically important problems such as the structural organization of multi-domain proteins and multi-protein assemblies look very promising.

Carbohydrates↗
PubMed2001

Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands.

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.

Amino Sugars↗
PubMed2000

Rapid determination of protein folds using residual dipolar couplings.

Over the next few years, various genome projects will sequence many new genes and yield many new gene products. Many of these products will have no known function and little, if any, sequence homology to existing proteins. There is reason to believe that a rapid determination of a protein fold, even at low resolution, can aid in the identification of function and expedite the determination of structure at higher resolution. Recently devised NMR methods of measuring residual dipolar couplings provide one route to the determination of a fold. They do this by allowing the alignment of previously identified secondary structural elements with respect to each other. When combined with constraints involving loops connecting elements or other short-range experimental distance information, a fold is produced. We illustrate this approach to protein fold determination on (15)N-labeled Eschericia coli acyl carrier protein using a limited set of (15)N-(1)H and (1)H-(1)H dipolar couplings. We also illustrate an approach using a more extended set of heteronuclear couplings on a related protein, (13)C, (15)N-labeled NodF protein from Rhizobium leguminosarum.

Acyl Carrier Protein↗
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↗