Prediction of the three-dimensional structure of proteins using the electrostatic screening model and hierarchic condensation

We describe a method for predicting the three-dimensional (3-D) structure of proteins from their sequence alone. The method is based on the electrostatic screening model for the stability of the protein main-chain conformation. The free energy of a protein as a function of its conformation is obtained from the potentials of mean force analysis of high-resolution x-ray protein structures. The free energy function is simple and contains only 44 fitted coefficients. The minimization of the free energy is performed by the torsion space Monte Carlo procedure using the concept of hierarchic condensation. The Monte Carlo minimization procedure is applied to predict the secondary, super-secondary, and native 3-D structures of 12 proteins with 28-110 amino acids. The 3-D structures of the majority of local secondary and super-secondary structures are predicted accurately. This result suggests that control in forming the native-like local structure is distributed along the entire protein sequence. The native 3-D structure is predicted correctly for 3 of 12 proteins composed mainly from the alpha-helices. The method fails to predict the native 3-D structure of proteins with a predominantly beta secondary structure. We suggest that the hierarchic condensation is not an appropriate procedure for simulating the folding of proteins made up primarily from beta-strands. The method has been proved accurate in predicting the local secondary and super-secondary structures in the blind ab initio 3-D prediction experiment.     

Phage display of proteins

In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature.     

A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis

The crystal structure of bovine neurophysin-II in its liganded state (Chen et al. [1991] Proc. Natl. Acad. Sci. USA 88, 4240-4244) indicates that the 1-6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer-monomer interface. Cleavage of the 1-6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1-6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des-1-6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1-6 sequence differed in liganded and unliganded states. Additionally, the unliganded des-1-6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des-1-6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des-1-6 protein, as evidenced by signals from an internal dimerization-sensitive alpha-proton. Thus, the 1-6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1-6 protein with an internal clip after binding site residue Glu-47, the clip presumably breaking the short 3,10 helix that most directly connects the interface to the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des-1-6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1-6 sequence. A model is proposed in which the 1-6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self-association.     

The Oms66 (p66) protein is a Borrelia burgdorferi porin

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.     

Residue-residue mean-force potentials for protein structure recognition

We present two new sets of energy functions for protein structure recognition, given the primary sequence of amino acids along the polypeptide chain. The first set of potentials is based on the positions of alpha- and the second on positions of beta- and alpha-carbon atoms of amino acid residues. The potentials are derived using a theory of Boltzmann-like statistics of protein structure. The energy terms incorporate both long-range interactions between residues remote along a chain and short-range interactions between near neighbors. Distance dependence is approximated by a piecewise constant function defined on intervals of equal size. The size of the interval is optimized to preserve as much detail as possible without introducing excessive error due to limited statistics. A database of 214 non-homologous proteins was used both for the derivation of the potentials, and for the 'threading' test originally suggested by Hendlich et al. (1990) J. Mol. Biol., 216, 167-180. Special care is taken to avoid systematic error in this test. For threading, we used 100 non-homologous protein chains of 60-205 residues. The energy of each of the native structures was compared with the energy of 43,000 to 19,000 alternative structures generated by threading. Of these 100 native structures, 92 have the lowest energy with alpha-carbon-based potentials and, even more, 98 of these 100 structures, have the lowest energy with the beta- and alpha-carbon based potentials.     

Multiplane transesophageal Doppler echocardiographic measurements of the velocity profile in the human pulmonary artery

A new method to detect remote relationships between protein sequences and known three-dimensional structures based on direct energy calculations and without reliance on statistics has been developed. The likelihood of a residue to occupy a given position on the structural template was represented by an estimate of the stabilization free energy made after explicit prediction of the substituted side chain conformation. The profile matrix derived from these energy values and modified by increasing the residue self-exchange values successfully predicted compatibility of heat-shock protein and globin sequences with the three-dimensional structures of actin and phycocyanin, respectively, from a full protein sequence databank search. The high sensitivity of the method makes it a unique tool for predicting the three-dimensional fold for the rapidly growing number of protein sequences.     

Physiological red blood cell kinetic model to explain the apparent discrepancy between adenosine breakdown inhibition and nucleoside transporter occupancy of draflazine

We present a fast method for finding optimal parameters for a low-resolution (threading) force field intended to distinguish correct from incorrect folds for a given protein sequence. In contrast to other methods, the parameterization uses information from >10(7) misfolded structures as well as a set of native sequence-structure pairs. In addition to testing the resulting force field's performance on the protein sequence threading problem, results are shown that characterize the number of parameters necessary for effective structure recognition.     

Reaching the global minimum in docking simulations: a Monte Carlo energy minimization approach using Bezier splines

The docking problem faces two major challenges: the global optimization of a multivariable function, such as the energy, and the ability to discriminate between true and false positive results, i.e., native from nonnative structures based on the input energy function. Among all energy evaluation tools, only a local energy-minimization method using an accurate enough potential function is able to discriminate between native and nonnative structures. To meet these requirements, a Monte Carlo with energy-minimization method has been incorporated into a new ECEPP/3 docking program. The efficiency of the simulation results from the use of an energy-grid technique based on Bezier splines and from a simplification of the receptor by switching on the energy of only important residues of the active site. Simulations of a thrombin-inhibitor complex show that the global minimum of the energy function was reached in every independent run within less than 3 min of time on an IBM RX 6000 computer. For comparison, 10 standard independent Monte Carlo simulations with 10(6) steps in each were carried out. Only three of them led to a conformation close to the x-ray structure. The latter simulations required an average of 24 min and about 10 hr with and without the grid, respectively. Another important result is that the Bezier spline technique not only speeds up the calculation by reducing the number of operations during the energy evaluation but also helps in reaching the global minimum by smoothing out the potential energy surface.     

New insights into p53 function from structural studies

Recent structural analysis of p53 has greatly enhanced our understanding of the biochemical activities of this protein by presenting us with a detailed picture of the chemical groups in the protein that are involved in protein stability, conformation and functional interactions. The current structures form the basis for the design of potential therapeutics which could, for example, revert a DNA-binding mutant back to a DNA-binding competent conformation. The structure of the tet domain forms the basis for designing an active therapeutic p53 with an oligomerization domain which would not cross react with a DNA-binding mutant p53. However, as useful as these structures have been in providing insight into the structure/function relationship for p53, a complete understanding of this protein awaits more detailed information on the full-length protein. In this respect, one of the most useful roles for future structural studies will be to help identify the nature of the conformational transition between latent and active p53, and how it can be modulated.     

Folding propensities of peptide fragments of myoglobin

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

TESS: a geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites

It is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X-ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O-His-O "catalytic triad" and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions.     

Sticky egyptians: a technique for assembling genes encoding constrained peptides of variable length

Naturally occurring peptides, such as those produced by the poisonous marine snails of the genus Conus , have the ability to form tight, highly specific molecular interactions. The rigidity of the peptide framework which promotes these interactions is usually maintained by disulphide bonds, and it seems that the overall main chain conformation (or fold) of the peptide is determined by its length and the sequence distribution of the pairs of cysteine residues participating in these bonds. The fold of the peptide in turn is largely responsible for its shape. Since highly effective molecular interactions occur between species complementary in shape, we reasoned that peptides with the greatest potential in therapy or diagnosis would be found in a library of shapes, those peptides with a shape complementary to a given target being identified, for example, by selection. As a first step towards constructing such a peptide shape library, we have developed a method for assembling DNA fragments which encode an even number of cysteine residues and which are of variable length. We describe this method here.     

Protein structure prediction by threading. Why it works and why it does not

We developed a novel Monte Carlo threading algorithm which allows gaps and insertions both in the template structure and threaded sequence. The algorithm is able to find the optimal sequence-structure alignment and sample suboptimal alignments. Using our algorithm we performed sequence-structure alignments for a number of examples for three protein folds (ubiquitin, immunoglobulin and globin) using both "ideal" set of potentials (optimized to provide the best Z-score for a given protein) and more realistic knowledge-based potentials. Two physically different scenarios emerged. If a template structure is similar to the native one (within 2 A RMS), then (i) the optimal threading alignment is correct and robust with respect to deviations of the potential from the "ideal" one; (ii) suboptimal alignments are very similar to the optimal one; (iii) as Monte Carlo temperature decreases a sharp cooperative transition to the optimal alignment is observed. In contrast, if the template structure is only moderately close to the native structure (RMS greater than 3.5 A), then (i) the optimal alignment changes dramatically when an "ideal" potential is substituted by the real one; (ii) the structures of suboptimal alignments are very different from the optimal one, reducing the reliability of the alignment; (iii) the transition to the apparently optimal alignment is non-cooperative. In the intermediate cases when the RMS between the template and the native conformations is in the range between 2 A and 3.5 A, the success of threading alignment may depend on the quality of potentials used. These results are rationalized in terms of a threading free energy landscape. Possible ways to overcome the fundamental limitations of threading are discussed briefly.     

De novo design of the hydrophobic core of ubiquitin

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Photoactive yellow protein: a structural prototype for the three-dimensional fold of the PAS domain superfamily

PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the approximately 50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical approximately 150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of approximately 125-150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.     

Monte Carlo simulations of protein folding. II. Application to protein A, ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338-352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 A from the native C alpha trace, and the best coordinate rms from crambin is 3.18 A. For ROP monomer, the lowest coordinate rms from equivalent C alpha s of ROP dimer is 3.65 A. Thus, for two simple helical proteins and a small alpha/beta protein, the ability to predict protein structure from sequence has been demonstrated.     

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.     

Conformational analysis of a chain reversal in alpha-chymotrypsin

A complete conformational analysis of the fold (Asp-Lys-Thr-Gly) (residues 35-38), and additional adjacent residues of alpha-chymotrypsin has been performed. A comparison of these findings with those of Lewis et al. (1) is made, and a discussion of the implications of protein-fold models is discussed. This particular residue sequence prefers to bend over maintaining a helical conformation. However, the bend conformation of the tetramer is different from that of the native bend. The native bend conformation is nearly realized when an additional residue of the native primary structure is added to each side of the tetramer. Early and late folding-sequence studies suggests that while the native fold is of low energy, there are fold-points along the primary structure which are more stable. The structural implications of this finding are discussed.     

Preservation of native conformation during aluminium-induced aggregation of tau protein

Aluminium exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27Al NMR spectroscopy. The CD studies revealed a five-fold increase in the observed elipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. 27Al NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits, the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for this process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.     

Nitric oxide synthase. Structural studies using anti-peptide antibodies

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681-4690]. Overall, they allow prediction of whether each peptide region will be an alpha-helix, a beta-strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies. These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (approximately 50%). Most of the selected anti-peptide antibodies were not able to cross-react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross-reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required.(ABSTRACT TRUNCATED AT 400 WORDS)