Rapid prediction of multi-dimensional NMR data sets

We present a computational environment for Fast Analysis of multidimensional NMR DAta Sets (FANDAS) that allows assembling multidimensional data sets from a variety of input parameters and facilitates comparing and modifying such ??in silico?? data sets during the various stages of the NMR data analysis. The input parameters can vary from (partial) NMR assignments directly obtained from experiments to values retrieved from in silico prediction programs. The resulting predicted data sets enable a rapid evaluation of sample labeling in light of spectral resolution and structural content, using standard NMR software such as Sparky. In addition, direct comparison to experimental data sets can be used to validate NMR assignments, distinguish different molecular components, refine structural models or other parameters derived from NMR data. The method is demonstrated in the context of solid-state NMR data obtained for the cyclic nucleotide binding domain of a bacterial cyclic nucleotide-gated channel and on membrane-embedded sensory rhodopsin II. FANDAS is freely available as web portal under WeNMR (http://www.wenmr.eu/services/FANDAS).     

Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein

Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.     

Rigid domains in proteins: An algorithmic approach to their identification

A rigid domain, defined here as a tertiary structure common to two or more different protein conformations, can be identified numerically from atomic coordinates by finding sets of residues, one in each conformation, such that the distance between any two residues within the set belonging to one conformation is the same as the distance between the two structurally equivalent residues within the set belonging to any other conformation. The distance between two residues is taken to be the distance between their respective α carbon atoms. With the methods of this paper we have found in the deoxy and oxy conformations of the human hemoglobin α₁β₁ dimer a rigid domain closely related to that previously identified by Baldwin and Chothia (J. Mol. Biol. 129:175–220,1979). We provide two algorithms, both using the difference-distance matrix, with which to search for rigid domains directly from atomic coordinates. The first finds all rigid domains in a protein but has storage and processing demands that become prohibitively large with increasing protein size. The second, although not necessarily finding every rigid domain, is computationally tractable for proteins of any size. Because of its efficiency we are able to search protein conformations recursively for groups of non-intersecting domains. Different protein conformations, when aligned by superimposing their respective domain structures; can be examined for structural differences in regions complementing a rigid domain. © 1995 Wiley-Liss, Inc.     

Evidence for allosteric effects on p53 oligomerization induced by phosphorylation

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)‐binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the “activating” C‐terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked‐immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild‐type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the “unfolded” conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.     

CADB: Conformation Angles DataBase of proteins

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/.     

A conformational switch in bacteriophage p22 portal protein primes genome injection

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.     

Continuous foaming for protein recovery: part I. Recovery of beta-casein

Foam separation is known to have potential for separation of biological molecules with a range of surface activities. A statistical study (factorial design) was carried out to establish the optimum operating conditions for the continuous foam separation of beta-casein. Maximum values of enrichment of beta-casein into the foam phase were found for low levels of initial feed protein concentration, gas flow rate, feed-flow rate, and high foam heights. Maximum values of protein recovery, were generally found at high levels of initial feed protein concentration, gas-flow rate, feed-flow rate, and low foam heights. The highest values obtained for enrichment and separation ratio were 54.7 and 181.3, respectively, with a simultaneous protein recovery of 62%; thus, illustrating the potential effectiveness of this technique. The effect of foaming on protein conformation is also important, and in this study protein structure was analyzed before and after foam separation experiments. Techniques used were: native polyacrylamide gel electrophoresis (PAGE), UV absorbance spectroscopy, circular dichroism, and fluorescence. Native PAGE showed no detectable changes in protein structure. However, absorbance scanning, fluorimetry, and circular dichroism revealed some conformational changes over a range of concentration effects.     

Molecular modelling of glycans: three-dimensional structure and protein fraction interaction. The example of rabbit sero-transferrin

On the basis of experimental data and of computer calculations using the Tripos 5.3 force field in order to examine the three-dimensional structures which are sterically feasible and the conformations which are energetically the most favourable, we have designed a program of molecular modelling of biantennary glycans of the N-acetyllactosaminic type (complex type). We demonstrate that, in absence of any interaction with the protein, a high number of glycan conformations exists which can be classified into five basic conformations, four of which have already been described. In fact, in addition to the Y-, T-, "bird" and "broken-wing" conformations, a "back-folded wing" conformation is energetically feasible. In contrast, the glycan linked to the protein is immobilized into only one conformation: the "broken-wing" conformation. Forming a bridge between the two lobes of the peptide chain, it probably contributes to the maintenance of the protein in a biologically active conformation.     

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete -helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.     

Lipid interaction converts prion protein to a PrPSc-like proteinase K-resistant conformation under physiological conditions

The conversion of prion protein (PrP) to the pathogenic PrPSc conformation is central to prion disease. Previous studies revealed that PrP interacts with lipids and the interaction induces PrP conformational changes, yet it remains unclear whether in the absence of any denaturing treatment, PrP-lipid interaction is sufficient to convert PrP to the classic proteinase K-resistant conformation. Using recombinant mouse PrP, we analyzed PrP-lipid interaction under physiological conditions and followed lipid-induced PrP conformational change with proteinase K (PK) digestion. We found that the PrP-lipid interaction was initiated by electrostatic contact and followed by hydrophobic interaction. The PrP-lipid interaction converted full-length alpha-helix-rich recombinant PrP to different forms. A significant portion of PrP gained a conformation reminiscent of PrPSc, with a PrPSc-like PK-resistant core and increased beta-sheet content. The efficiency for lipid-induced PrP conversion depended on lipid headgroup structure and/or the arrangement of lipids on the surface of vesicles. When lipid vesicles were disrupted by Triton X-100, PrP aggregation was necessary to maintain the lipid-induced PrPSc-like conformation. However, the PK resistance of lipid-induced PrPSc-like conformation does not depend on amyloid fiber formation. Our results clearly revealed that the lipid interaction can overcome the energy barrier and convert full-length alpha-helix-rich PrP to a PrPSc-like conformation under physiological conditions, supporting the relevance of lipid-induced PrP conformational change to in vivo PrP conversion.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Metal-dependent protein folding: metallation of metallothionein

Metallothionein (MT), a low molecular mass, cysteine-rich protein, is a model system for metal ion-directed folding due to its diverse metal binding properties. In this minireview, the current status of theoretical and experimental studies that have focused primarily on the initial metallation steps involving the metal-free, or apo, MT and divalent metals, Zn²⁺ and Cd²⁺ is described. Apo-MT has recently been reported to be present in the cell in quantities equal to that of the metallated protein, which might indicate a potential role for the protein in the absence of metals. Molecular mechanics-molecular dynamics (MM3/MD) calculations carried out on the demetallation of cadmium-coordinated MT isoform 1a indicate structural stability of the metal-free protein with significant retention of the backbone conformation imposed by the metal-thiolate clusters present in the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT starting from a linear strand did not possess any structural stability and can be described as a random coil conformation. Evidence for the sequence of metallation is discussed, together with current experimental data to support either a cooperative or sequential binding mechanisms.     

Conformation and stability of Sendai virus fusion protein

The conformation and stability of Sendai virus fusion (F) protein were studied by circular dichroism spectroscopy, and the protein predictive models of Chou and Fasman and Robson and Suzuki were used to elucidate the secondary structure of Sendai virus F protein. The F protein conformation is predicted to contain 33% alpha-helix, 53% beta-sheet and 15% beta-turn by the Chou and Fasman model, and 30% alpha-helix, 55% beta-sheet, 9% beta-turn and 7% random coil by the Robson and Suzuki model. C.d. studies of F protein purified in the presence of the non-ionic detergent, n-octylglucoside, indicated the presence of 49% alpha-helix and 31% beta-sheet at pH 7.0, 54% alpha-helix and 28% beta-sheet at pH 9.0 and 50% alpha-helix and 23% beta-sheet at pH 5.4. A small change in conformation of the protein occurred when the pH was titrated from 7.0 to 5.4 and from 7.0 to 9.0 and a more pronounced conformational change occurred when the pH was changed from 9.0 to 5.4. The F protein in 0.2% n-octylglucoside was resistant to denaturation by 4 M guanidine hydrochloride, the reducing agent 20 mM mercaptoethanol, and to increases in temperature from 5 to 80 degrees C. Monoclonal anti-F protein antibody showed an increased binding to whole virus when the pH was changed from 7.0 to 9.0. The antibody binding was decreased when the pH was shifted from 9.0 to 5.4 Maximum haemolytic activity was observed with virus that was preincubated at pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyses of the Sequence and Structural Properties Corresponding to Pentapeptide and Large Palindromes in Proteins

The analyses of 3967 representative proteins selected from the Protein Data Bank revealed the presence of 2803 pentapeptide and large palindrome sequences with known secondary structure conformation. These represent 2014 unique palindrome sequences. 60% palindromes are not associated with any regular secondary structure and 28% are in helix conformation, 11% in strand conformation and 1% in the coil conformation. The average solvent accessibility values are in the range between 0–155.28 Ų suggesting that the palindromes in proteins can be either buried, exposed to the solvent or share an intermittent property. The number of residue neighborhood contacts defined by interactions ≤ 3.2 Ǻ is in the range between 0–29 residues. Palindromes of the same length in helix, strand and coil conformation are associated with different amino acid residue preferences at the individual positions. Nearly, 20% palindromes interact with catalytic/active site residues, ligand or metal ions in proteins and may therefore be important for function in the corresponding protein. The average hydrophobicity values for the pentapeptide and large palindromes range between -4.3 to +4.32 and the number of palindromes is almost equally distributed between the negative and positive hydrophobicity values. The palindromes represent 107 different protein families and the hydrolases, transferases, oxidoreductases and lyases contain relatively large number of palindromes.     

The molecular structure of the receptor-ionophore complex at the neuromuscular junction.

A general theory of the molecular structure of receptors for transmitters based only on protein has been presented elsewhere (Smythies, 1974a,b). The acetylcholine receptor at the neuromuscular junction is postulated in particular to be based on a Kusnetsov-Ghokov grid with four sequencestwo “primary” chains A-x-B-cys-A-x-B where A = arg or lys and B = glu or phosphoser and two “secondary” chains of sequence -gly-x-gly-pro-x-ile-cys-asp-x- forming a symmetrical receptor cup of rectangular form. The present paper extends the model to include the gate over the adjacent ionophore (or “ion conductance modulator”: ICM) and the linking mechanism from receptor to gate. These are postulated to consist of a second Kusnetsov-Ghokov grid generated by a third “primary” chain along the side that covers the orifice to the ion conducting channel. The action of ACh is postulated to be to displace an hydrated Ca⁺⁺ ion from the receptor cup and to disrupt the AB rungs in the receptor grid. The middle primary chain then slides 14 Å and the AB links reform. This replaces a bulky amino acid pair normally blocking the ion channel by a less bulky amino acid pair and so hydrated ions can be transmitted. It is further postulated that snake neurotoxins (ACh blockers) in a specified conformation bind mainly to the ionophore grid and prevent the sliding filament mechanism from opening; whereas the snake “cardiotoxins” (ACh agonists)—in a specified conformation—bind to the same sliding filament mechanism in its “open” ionophore gate and prevent it being closed: and histrionicotoxin binds to the same open “gate” but blocks it physically. The hypothesis may rigorously be tested by experiment as it makes detailed predictions on the X-ray structure of the snake neurotoxins and cardiotoxins.     

Terminal residues in protein chains: residue preference, conformation, and interaction

The known protein structures have been analyzed to find out if there is any pattern in the type of residues used and their conformation at the two terminal positions of the polypeptide chains. While the N-terminal position is overwhelmingly occupied by Met (followed by Ala and Ser), the preference for the C-terminal is not as distinct, the residues with highest propensities being Lys, Arg, Gln, and Asn. Only one main-chain torsion angle, psi, can be defined for the N-terminal residue, which is found to be in the extended conformation due to a favorable electrostatic interaction between the charged amino group and the carbonyl oxygen atom. The distribution of the angle phi for the C-terminal residue, on the other hand, is not much different from that of the nonterminal residues. There are some differences in the distribution of the side-chain torsion angle chi1 of both the terminal residues from the general distribution. The terminal segments are generally flexible and there is a tendency for the more ordered residues to have lesser solvent exposure. About 40% of the terminal groups form a hydrogen bond with protein atoms--a slight preference is observed for the side-chain atoms (more than half of which belong to charged residues) over the main-chain ones. Although the terminal residues are not included in any regular secondary structure, the adjacent ones have a high preference to occur in the beta conformation. There is a higher chance of a beta-strand rather than an alpha-helix to start within the first 6 positions from the N-terminal end. It is suggested that the extended conformation observed for the N-terminal residue propagates along the chain leading to the formation of beta-strand. In the C-terminal end, on the other hand, as one moves upstream the alpha and beta structures are encountered in proportion similar to the average value for these structures in the database. The cleavage site of the zymogen structures has a conformation that can be retained by the N-terminal residue of the active enzyme.     

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.