Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy.

The structure and dynamics of the urea-denatured B1 immunoglobulin binding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 1H, 15N, and 13C assignments are obtained by means of sequential through-bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHN alpha coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. 15N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second beta-hairpin, the N-terminus of the alpha-helix, and the middle of the alpha-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (approximately 3 ns) and could range from about 6 ns in the case of one model to between 4 microseconds and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N- and C-termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.

The protein synthesis elongation factor 1A (eEF1A) is a multifunctional protein in eukaryotic cells. In maize (Zea mays L.) endosperm eEF1A co-localizes with actin around protein bodies, and its accumulation is highly correlated with the protein-bound lysine (Lys) content. We purified eEF1A from maize kernels by ammonium sulfate precipitation, ion-exchange, and chromatofocusing. The identify of the purified protein was confirmed by microsequencing of an endoproteinase glutamic acid-C fragment and by its ability to bundle actin. Using purified eEF1A as a standard, we found that this protein contributes 0.4% of the total protein in W64A+ endosperm and approximately 1% of the protein in W64Ao2. Because eEF1A contains 10% Lys, it accounts for 2.2% of the total Lys in W64A+ and 2.3% of the Lys in W64Ao2. However, its concentration predicts 90% of the Lys found in endosperm proteins of both genotypes, indicating that eEF1A is a key component of the group of proteins that determines the nutritional quality of the grain. This notion is further supported by the fact that in floury2, another high-Lys mutant, the content of eEF1A increases with the dosage of the floury2 gene. These data provide the biochemical basis for further investigation of the relationship between eEF1A content and the nutritional quality of cereals.     

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.     

The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.     

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.     

Nuclear magnetic resonance study of the solution structure of alpha 1-purothionin. Sequential resonance assignment, secondary structure and low resolution tertiary structure

The solution structure of the 45-residue plant protein, alpha 1-purothionin, is investigated by nuclear magnetic resonance (n.m.r.) spectroscopy. Using a combination of two-dimensional n.m.r. techniques to demonstrate through-bond and through-space (less than 5 A) connectivities, the 1H n.m.r. spectrum of alpha 1-purothionin is assigned in a sequential manner. The secondary structure elements are then delineated on the basis of a qualitative interpretation of short-range nuclear Overhauser effects (NOE) involving the NH, C alpha H and C beta H protons. There are two helices extending from residues 10 to 19 and 23 to 28, two short beta-strands from residues 3 to 5 and 31 to 34 which form a mini anti-parallel beta-sheet, and five turns. In addition, a number of long-range NOE connectivities are assigned and a low resolution tertiary structure is proposed.     

Application of molecular dynamics with interproton distance restraints to three-dimensional protein structure determination. A model study of crambin

The applicability of restrained molecular dynamics for the determination of three-dimensional protein structures on the basis of short interproton distances (less than 4 A) that can be realistically determined from nuclear magnetic resonance measurements in solution is assessed. The model system used is the 1.2 A resolution crystal structure of the 46 residue protein crambin, from which a set of 240 approximate distance restraints, divided into three ranges (2.5 +/- 0.5, 3.0+0.5(-1.0) and 4 +/- 1 A), is derived. This interproton distance set comprises 159 short-range ([i-j] less than or equal to 5) and 56 ([i-j] greater than 5) long-range inter-residue distances and 25 intra-residue distances. Restrained molecular dynamics are carried out using a number of different protocols starting from two initial structures: a completely extended beta-strand; and an extended structure with two alpha-helices in the same positions as in the crystal structure (residues 7 to 19, and 23 to 30) and all other residues in the form of extended beta-strands. The root-mean-square (r.m.s.) atomic differences between these two initial structures and the crystal structure are 43 A and 23 A, respectively. It is shown that, provided protocols are used that permit the secondary structure elements to form at least partially prior to folding into a tertiary structure, convergence to the correct final structure, both globally and locally, is achieved. The r.m.s. atomic differences between the converged restrained dynamics structures and the crystal structure range from 1.5 to 2.2 A for the backbone atoms and from 2.0 to 2.8 A for all atoms. The r.m.s. atomic difference between the X-ray structure and the structure obtained by first averaging the co-ordinates of the converged restrained dynamics structures is even smaller: 1.0 A for the backbone atoms and 1.6 A for all atoms. These results provide a measure with which to judge future experimental results on proteins whose crystal structures are unknown. In addition, from an examination of the dynamics trajectories, it is shown that the convergence pathways followed by the various simulations are different.     

The three-dimensional structure of alpha1-purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The determination of the three-dimensional solution structure of α1-purothionin using a combination of metric matrix distance geometry and restrained molecular dynamics calculations based on n.m.r. data is presented. The experimental data comprise complete sequence-specific proton resonance assignments, a set of 310 approximate interproton distance restraints derived from nuclear Overhauser effects, 27 Ø backbone torsion angle restraints derived from vicinal coupling constants, 4 distance restraints from hydrogen bonds and 12 distance restraints from disulphide bridges. The average atomic rms difference between the final nine converged structures and the mean structure obtained by averaging their coordinates is 1.5 ± 0.1 å for the backbone atoms and 2.0 ± 0.1 å for all atoms. The overall shape of α1-purothionin is that of the capital letter L, similar to that of crambin, with the longer arm comprising two approximately parallel α-helices and the shorter arm a strand and a mini anti-parallel β sheet.