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PubMed2000

Binding of tropomyosin-troponin to actin increases filament bending stiffness.

Rheologic measurements show that the association of tropomyosin-troponin with actin filaments is responsible for the reduction of the internal chain dynamic and increase in the mechanical rigidity of actin filaments. Basing calculations on the linear relation between the plateau modulus, G(N)('), and bending modulus, kappa, I find that tropomyosin-troponin at r(AT) = 7 increases actin filament stiffness by approximately 50%. This is confirmed by dynamic light scattering. Further increases are observed at rising F-actin and constant tropomyosin-troponin concentrations. Tropomyosin-troponin also delays actin assembly and subsequent network formation and increases filament stiffness over time.

Actin Cytoskeleton↗

PubMed2000

Glycation of aspartate aminotransferase and conformational flexibility.

Glycation of proteins alters biological function and changes cellular processes. Our study investigated the conformational changes that accompany glycation using the cardiac aspartate aminotransferase (cAAT). We examined the effects of brief and prolonged exposure of cAAT to glyceraldehyde (Glyc) and ribose 5-phosphate (R5P). When cAAT was briefly incubated (3.5 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, cAAT activity and 1-anilinonaphthalene 8-sulfonate (ANS) binding increased relative to control. After prolonged incubation (64 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, activity and ANS binding decreased relative to control. Furthermore, upon prolonged incubation of cAAT with 500 microM Glyc (14.5 h) or 2 mM R5P (64.25 h) at 37 degrees C, the denaturation curves shifted to the right relative to control. We conclude that upon brief incubation with Glyc and R5P, cAAT exhibited a more open and flexible structure and upon prolonged incubation, a more rigid structure.

Anilino Naphthalenesulfonates↗

PubMed2001

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.

Amino Acid Sequence↗

PubMed2001

A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.

Carbon-Oxygen Lyases↗

PubMed1997

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.

3-Isopropylmalate Dehydrogenase↗

PubMed1998

Conformational flexibility in a highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin and antibody binding.

The G-H loop of foot-and-mouth disease virus VP1 protein is a highly mobile peptide, that extends from the capsid surface and that in native virions is invisible by X-ray crystallography. In serotype C, this segment contains a hypervariable region with several continuous, overlapping, B-cell epitopes that embrace the conserved Arg-Gly-Asp (RGD) cell attachment motif. The solvent-exposed positioning of this peptide by selective insertion into different structural frameworks of E. coli beta-galactosidase, generates a spectrum of antigenic variants which react distinctively with a panel of anti-VP1 monoclonal antibodies and exhibit different efficiencies as cell ligands. The cell attachment efficiency is much less restricted by the different positioning of the viral segment at the insertion sites. A molecular model of an inserted stretch reveals a highest flexibility of the RGD tripeptide segment compared with the flanking sequences, that could allow a proper accommodation to integrin receptors even in poorly antigenic conformations. The non-converging structural requirements for RGD-mediated integrin binding and antibody recognition, explains the dynamism of the generation of neutralisation-resistant antigenic variants in the viral quasi-species, arising from a conformational space of integrin-binding competent peptides. This might be of special relevance for foot-and-moth disease virus evolution, since unlike in other picornaviruses, the cell binding motif and the major neutralising B-cell epitopes overlap in a solvent-exposed peptide accessible to the host immune system, in a virion lacking canyons and similar hiding structures.

Amino Acid Sequence↗

PubMed1998

Engrailed homeodomain-DNA complex at 2.2 A resolution: a detailed view of the interface and comparison with other engrailed structures.

We report the 2.2 A resolution structure of the Drosophila engrailed homeodomain bound to its optimal DNA site. The original 2.8 A resolution structure of this complex provided the first detailed three-dimensional view of how homeodomains recognize DNA, and has served as the basis for biochemical studies, structural studies and molecular modeling. Our refined structure confirms the principal conclusions of the original structure, but provides important new details about the recognition interface. Biochemical and NMR studies of other homeodomains had led to the notion that Gln50 was an especially important determinant of specificity. However, our refined structure shows that this side-chain makes no direct hydrogen bonds to the DNA. The structure does reveal an extensive network of ordered water molecules which mediate contacts to several bases and phosphates (including contacts from Gln50), and our model provides a basis for detailed comparison with the structure of an engrailed Q50K altered-specificity variant. Comparing our structure with the crystal structure of the free protein confirms that the N and C termini of the homeodomain become ordered upon DNA-binding. However, we also find that several key DNA contact residues in the recognition helix have the same conformation in the free and bound protein, and that several water molecules also are "preorganized" to contact the DNA. Our structure helps provide a more complete basis for the detailed analysis of homeodomain-DNA interactions.

Crystallography, X-Ray↗

PubMed2000

Pulling chromatin fibers: computer simulations of direct physical micromanipulations.

A low-resolution molecular model, which combines the known mechanical properties of protein-free DNA with the accumulating picture of chromatosome structure, has been developed to account for the stretching of single chromatin fibers by an imposed external force. Force-extension characteristics of sets of chains accumulated by Monte Carlo sampling are consistent with recently observed findings in the non-destructive regime (<20 pN imposed force), where the structure of the chromatosome remains intact. The correspondence between simulation and the relaxation phase of the experiment limits the equilibrium entry-exit angle of linker DNA on the chromatosome to W=50(+/-10) degrees and the effective DNA linker length to L(eff)=40(+/-5) bp. The computed force-extension characteristics are relatively insensitive to other parameters of the model, precluding their accurate estimation. The introduction of an attractive potential between closely spaced nucleosomes reproduces the added initial resistance of single fibers to extension at high salt conditions. The consideration of elastic linkers also improves the fitting of assorted classical measurements of unstressed chromatin structure in solution. The overall picture of chromatin that emerges is an irregular, fluctuating, three-dimensional, zig-zag structure with intact, mechanically stable chromatosome units and deformable linkers. The modeled fiber undergoes large-scale configurational rearrangements without significant perturbation of the constituent chromatosome beads, collapsing into a highly condensed form in response to small (<2kT) inter-nucleosomal attractions.

Calibration↗

PubMed1999

De novo protein design. II. Plasticity in sequence space.

It is generally accepted that many different protein sequences have similar folded structures, and that there is a relatively high probability that a new sequence possesses a previously observed fold. An indirect consequence of this is that protein design should define the sequence space accessible to a given structure, rather than providing a single optimized sequence. We have recently developed a new approach for protein sequence design, which optimizes the complete sequence of a protein based on the knowledge of its backbone structure, its amino acid composition and a physical energy function including van der Waals interactions, electrostatics, and environment free energy. The specificity of the designed sequence for its template backbone is imposed by keeping the amino acid composition fixed. Here, we show that our procedure converges in sequence space, albeit not to the native sequence of the protein. We observe that while polar residues are well conserved in our designed sequences, non-polar amino acids at the surface of a protein are often replaced by polar residues. The designed sequences provide a multiple alignment of sequences that all adopt the same three-dimensional fold. This alignment is used to derive a profile matrix for chicken triose phosphate isomerase, TIM. The matrix is found to recognize significantly the native sequence for TIM, as well as closely related sequences. Possible application of this approach to protein fold recognition is discussed.

Amino Acid Sequence↗

PubMed1999

Tumor suppressor INK4: comparisons of conformational properties between p16(INK4A) and p18(INK4C).

The INK4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15(INK4B), p16(INK4A), p18(INK4C), and p19(INK4D). While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear (1)H-(15)N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/(2)H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18<p16</=p15. A subset of very slowly exchanging residues (about 19 in total) was identified in p18, including 16 residues in the region of the fourth ankyrin repeat, probably as a result of a stabilizing effect by the extra ankyrin repeat. Thus, while INK4 proteins may have similar low thermodynamic stability as well as limited flexibility on the pico- to nanosecond time-scale, they display pronounced differences in the conformational flexibility on the time-scale of minutes to hours. Further analyses suggested that differences in H/(2)H exchange rates reflect differences in the kinetic stability of the INK4 proteins, which in turn is related to differences in the aggregation tendency.

Ankyrins↗

PubMed2000

Sequence-dependent DNA structure: dinucleotide conformational maps.

We have used a computational model to calculate the potential energy surface for dinucleotide steps in double helical DNA as a function of the two principal degrees of freedom, slide and shift. By using a virtual bond to model the constraints imposed by the sugar-phosphate backbone, twist, roll, tilt and rise can be simultaneously optimised for any given values of slide and shift. Thus we have been able to construct complete conformational maps for all step types. For some steps, the maps agree well with experimental data from X-ray crystal structures, but other steps appear to be strongly perturbed by the effects of context (conformational coupling with the neighbouring steps). The optimised values of twist and roll show sequence-dependent variations consistent with the crystal structure data. The conformational maps allow us to construct adiabatic paths, and hence calculate the flexibility of each step with respect to slide and shift. Again the results agree well with the available experimental assignments of flexibility: YR steps, CA/TG and CG, are the most flexible and RR steps, such as AA, the least flexible.

Base Pairing↗

PubMed2000

Sequence-dependent DNA structure: tetranucleotide conformational maps.

A database of X-ray crystal structures of double helical DNA oligomers has been used to analyse the role of the sugar-phosphate backbone in coupling the conformational properties of neighbouring dinucleotide steps. The base step parameters which are most strongly coupled to the backbone degrees of freedom are slide and shift, and these are the two dinucleotide step parameters which show strong correlations along a sequence: the value of slide follows the values in the neighbouring steps, whereas shift tends to alternate. This conformational coupling is mediated by the shared furanose rings at the step junctions: a change in the value of slide causes a change in the mean value of the same strand 3' and 5'-chi torsion angle, and a change in the mean value of the 3' and 5' sugar pseudo-rotation phase angle, P; a change in the value of shift causes a difference between the same strand 3' and 5'-chi in A-DNA and a difference between the 3' and 5'-P in B-DNA. We have used a database of tetranucleotide X-ray crystal structures to parameterise a simple model for the coupling of slide and shift. Using this junction model together with our dinucleotide step potential energy maps described previously, we can in principle calculate the structure of any DNA oligomer. The parameterisation indicates that the rotational step parameters are accurate to within 5 degrees, and the translational step parameters are accurate to within 0.5 A. The model has been used to study the potential energy surfaces of all possible tetranucleotide sequences, and the calculations agree well with the experimental data from X-ray crystal structures. Some dinucleotide steps are context independent (AA/TT, AT and TA), because the conformational properties of all possible neighbouring steps are compatible. When the conformational properties of the neighbours are not compatible, the behaviour of a step cannot be understood at the dinucleotide level. Thus the conformations of CG, GC and GG/CC are all strongly context dependent. The remaining mixed sequence steps show weakly context-dependent behaviour. The approach allows the calculation of the relative stability and flexibility of tetranucleotide sequences, and the results indicate why TATA is used as an origin of replication. Clear predictions are made about sequences which have not yet been characterised crystallographically. In particular, poly(CCA).poly(TGG) is predicted to have an unusual structure which lies between the C and D-DNA polymorphs.

Base Pairing↗

PubMed2000

Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles.

Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequence-dependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has first been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone tail-dependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series.

Acetylation↗

PubMed2000

NMR solution structure of the inserted domain of human leukocyte function associated antigen-1.

The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primarily via the inserted domain (I-domain) in the alpha-subunit. The activation of LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disagreement in the literature over the respective roles of conformational changes in the I-domain and of divalent cations (Mg(2+), Mn(2+)) in the activation of LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal alpha-helix; this change was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg(2+) complex of the I-domain of LFA-1 has been determined by NMR methods, using a model-based approach to nuclear Overhauser enhancement spectroscopy peak assignment. The protein adopts the same structure in solution as that of the published I-domain X-ray structures, but the C-terminal region, where the X-ray structures are most different from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consisting of breathing or segmental motion of the helix. The conformational diversity seen in the various X-ray structures could be explained as a result of the inherent flexibility of this C-terminal region and as a result of crystal contacts. Our NMR data are consistent with a model where the C-terminal helix has the potential flexibility to take up alternative conformations, for example, in the presence and absence of the intercellular adhesion molecule ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for the ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand and by modulation of the charge of the binding surface.

Amino Acid Sequence↗

PubMed2000

Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.

Amino Acid Sequence↗

PubMed2000

Conformational flexibility of B-DNA at 0.74 A resolution: d(CCAGTACTGG)(2).

The affinity and specificity of a ligand for its DNA site is a function of the conformational changes between the isolated and complexed states. Although the structures of a hydroxypyrrole-imidazole-pyrrole polyamide dimer with 5'-CCAGTACTGG-3' and the trp repressor recognizing the sequence 5'-GTACT-3' are known, the baseline conformation of the DNA site would contribute to our understanding of DNA recognition by these ligands. The 0.74 A resolution structure of a B-DNA double helix, 5'-CCAGTACTGG-3', has been determined by X-ray crystallography. Six of the nine phosphates, two of four bound calcium ions and networks of water molecules hydrating the oligonucleotide have alternate conformations. By contrast, nine of the ten bases have a single, unique conformation with hydrogen atoms visible in most cases. The polyamide molecules alter the geometry of the phosphodiester backbone, and the water molecules mediating contacts in the trp repressor/operator complex are conserved in the unliganded DNA. Furthermore, the multiple conformational states, ions and hydration revealed by this ultrahigh resolution structure of a B-form oligonucleotide are potentially general considerations for understanding DNA-binding affinity and specificity by ligands.

Bacterial Proteins↗

PubMed2000

Hyperstable stacked-disk structure of tobacco mosaic virus protein: electron cryomicroscopy image reconstruction related to atomic models.

The stacked disk aggregate of tobacco mosaic virus protein is an intriguing object due to its high degree of stability, in spite of indications that the aggregate is held together to a great extent by water-mediated interactions between adjacent protein rings. Here, we present a set of models that were constructed using the atomic coordinates of the four-layer aggregate, and compare these with a three-dimensional reconstruction of the stacked disk obtained by means of cryoelectron microscopy and helical image processing. The comparison of the four possible models of the stacked disk with the data shows that there is a better correlation of the data with the left-handed model built from the A-A ring pair coordinates than with the two models involving the A-B ring pair, or with the right-handed model of the A-A ring pair. This establishes that the packing of the protein subunits in the stacked disk is different from that previously believed. We also note some differences between the observed structure and A-A ring pair model in the region of the flexible loop at small radius that might be an indication of conformational differences that give rise to the stability of the aggregate.

Capsid↗

PubMed2000

Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions.

To find motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a database of transmembrane domains. Our analysis was performed with a novel formalism, which we call TMSTAT, for exactly calculating the expectancies of all pairs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of finite length in short segments. We found that the number of significantly over and under-represented pairs and triplets was much greater than the random expectation. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i+4 found in association with large aliphatic residues (Ile, Val and Leu) at neighboring positions (i.e. i+/-1 and i+/-2). The most over-represented pair is formed by two glycine residues at i and i+4 (GxxxG, 31.6 % above expectation, p<1x10(-33)) and it is strongly associated with the neighboring beta-branched residues Ile and Val. In fact, the GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identified using TOXCAT, an in vivo selection system for transmembrane oligomerization motifs. In conjunction with these experimental observations, our results highlight the importance of the GxxxG+beta-branched motif in transmembrane helix-helix interactions. In addition, the special role for the beta-branched residues Ile and Val suggested here is consistent with the hypothesis that residues with constrained rotameric freedom in helical conformation might reduce the entropic cost of folding in transmembrane proteins. Additional material is available at http://engelman.csb.yale. edu/tmstat and http://bioinfo.mbb.yale. edu/tmstat.

Amino Acid Motifs↗