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F E Cohen

Publications and source records attributed to F E Cohen.

At least 91 records · Page 5Linked to original sources
PubMed1997

Identification of candidate proteins binding to prion protein.

Prion diseases are disorders of protein conformation that produce neurodegeneration in humans and animals. Studies of transgenic (Tg) mice indicate that a factor designated protein X is involved in the conversion of the normal cellular prion protein (PrPC) into the scrapie isoform (PrPSc); protein X appears to interact with PrPC but not with PrPSc. To search for PrPC binding proteins, we fused PrP with alkaline phosphatase (AP) to produce a soluble, secreted probe. PrP-AP was used to screen a lambdagt11 mouse brain cDNA library, and six clones were isolated. Four cDNAs are novel while two clones are fragments of Nrf2 (NF-E2 related factor 2) transcription factor and Aplp1 (amyloid precursor-like protein 1). The observation that PrP binds to a member of the APP (amyloid precursor protein) gene family is intriguing, in light of possible relevance to Alzheimer's disease. Four of the isolated clones are expressed preferentially in the mouse brain and encode a similar motif.

Amino Acid Sequence↗
PubMed1997

Selective neuronal targeting in prion disease.

The pattern of scrapie prion protein (PrP(Sc)) accumulation in the brain is different for each prion strain. We tested whether the PrP(Sc) deposition pattern is influenced by the Asn-linked oligosaccharides of PrP(C) in transgenic mice. Deletion of the first oligosaccharide altered PrP(C) trafficking and prevented infection with two prion strains. Deletion of the second did not alter PrP(C) trafficking, permitted infection with one prion strain, and had a profound effect on the PrP(Sc) deposition pattern. Our data raise the possibility that glycosylation can modify the conformation of PrP(C). Glycosylation could affect the affinity of PrP(C) for a particular conformer of PrP(Sc), thereby determining the rate of nascent PrP(Sc) formation and the specific patterns of PrP(Sc) deposition.

Animals↗
PubMed1997

The prion folding problem.

Prion diseases are neurodegenerative disorders in which dramatic conformational change in the structure of the prion protein is the fundamental event. This structural transition involves the loss of substantial alpha-helical content and the acquisition of beta-sheet structure. A convergence of recent biological and structural studies argues that the mechanism underlying the prion diseases is truly unprecedented.

Biopolymers↗
PubMed1997

Kinetics and mechanism of amyloid formation by the prion protein H1 peptide as determined by time-dependent ESR.

BACKGROUND: Peptides derived from three of four putative alpha-helical regions of the prion protein (PrP) form amyloid in solution. These peptides serve as models for amyloidogenesis and for understanding the alpha helix -->beta strand conformational change that is responsible for the development of disease. Kinetic studies of amyloid formation usually rely on the detection of fibrils. No study has yet explored the rate of monomer peptide uptake or the presence of nonfibrillar intermediate species. We present a new electron spin resonance (ESR) method for probing the kinetics of amyloid formation. A spin label was covalently attached to a highly amyloidogenic peptide and kinetic trials were monitored by ESR. RESULTS: Electron microscopy shows that the spin-labeled peptide forms amyloid, and ESR reveals the kinetic decay of free peptide monomer during amyloid formation. The combination of electron microscopy and ESR suggests that there are three kinetically relevant species: monomer peptide, amyloid and amorphous aggregate (peptide aggregates devoid of fibrils or other structures with long-range order). A rather surprising result is that amyloid formation requires the presence of this amorphous aggregate. This is particularly interesting because PrPSc, the form of PrP associated with scrapie, is often found as an aggregate and amyloid formation is not a necessary component of prion replication or pathogenesis. CONCLUSIONS: Kinetic analysis of the time-dependent data suggests a model whereby the amorphous aggregate has a previously unsuspected dual role: it releases monomer into solution and also provides initiation sites for fibril growth. These findings suggest that the beta-sheet-rich PrPSc may be stabilized by aggregation.

Amyloid↗
PubMed1997

Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2), Asilomar, California, December 13-16, 1996.

In most fields of scientific endeavor, the outcomes of important experiments are not always known before the experiments are performed. But in protein structure prediction, algorithms are usually developed and tested in situations where the answers are known. In December 1996, the Second Meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2) was held in Asilomar, California to rectify this situation: protein sequences were provided in advance for which the experimental structure had not yet been published. Over 70 research groups provided bona fide predictions on 42 targets in four categories: comparative or 'homology' modeling, fold recognition or 'threading', ab initio structure predictions, and docking predictions. Since the previous CASP meeting in 1994, the role of fold recognition in structure prediction has increased enormously with the largest number of groups participating in this category. In this review, we highlight some of the important developments and give at least a qualitative sense of what kind of methods produced some of the better predictions.

Computer Simulation↗
PubMed1997

Chiral N-substituted glycines can form stable helical conformations.

BACKGROUND: Short sequence-specific heteropolymers of N-substituted glycines (peptoids) have emerged as promising tools for drug discovery. Recent work on medium-length peptoids containing chiral centers in their sidechains has demonstrated the existence of stable chiral conformations in solution. In this report, we explore the conformational properties of these N alpha chiral peptoids by molecular mechanics calculations and we propose a model for the solution conformation of an octamer of (S)-N-(1-phenylethyl)glycine. RESULTS: Molecular mechanics calculations indicate that the presence of N-substituents in which the N alpha carbons are chiral centers has a dramatic impact on the available backbone conformations. These results are supported by semi-empirical quantum mechanical calculations and coincide qualitatively with simple steric considerations. They suggest that an octamer of (S)-N-(1-phenylethyl)glycine should form a right-handed helix with cis amide bonds, similar to the polyproline type I helix. This model is consistent with circular dichorism studies of these molecules. CONCLUSIONS: Peptoid oligomers containing chiral centers in their sidechains present a new structural paradigm that has promising implications for the design of stably folded molecules. We expect that their novel structure may provide a scaffold to create heteropolymers with useful functionality.

Amino Acid Substitution↗
PubMed1997

Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an alpha-helix.

Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously developed fatal CNS illnesses similar to neuronal storage diseases. Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with the human counterpart of this malady will be found remains to be determined.

Animals↗
PubMed1997

A predicted consensus structure for the C terminus of the beta and gamma chains of fibrinogen.

A secondary structure has been predicted for the C termini of the fibrinogen beta and gamma chains from an aligned set of homologous protein sequences using a transparent method that extracts conformational information from patterns of variation and conservation, parsing strings, and patterns of amphiphilicity. The structure is modeled to form two domains, the first having a core parallel sheet flanked on one side by at least two helices and on the other by an antiparallel amphiphilic sheet, with an additional helix connecting the two sheets. The second domain is built entirely from beta strands.

Amino Acid Sequence↗
PubMed1997

A predicted consensus structure for the N-terminal fragment of the heat shock protein HSP90 family.

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment).

Algorithms↗
PubMed1996

Recombinant scrapie-like prion protein of 106 amino acids is soluble.

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.

Base Sequence↗
PubMed1996

Multiple sequence information for threading algorithms.

Threading algorithms attempt to solve the inverse protein folding problem: given a group of structures and a sequence, identify the structure that is most compatible with this sequence. A recent study of this class of algorithms by S. J. Wodak and colleagues suggests that while threading algorithms are capable of recognizing many folding motifs, their performance in truly blind predictions is disappointing, and the underlying alignments upon which the selections are based are frequently errant. To help overcome this problem we have developed a Test of Optimal Mutagenesis algorithm (TOM) that exploits information inherent in the variation between several homologues in a multiple sequence alignment. This information is used to help select the correct structural motif for the sequence from a database of known structures. A total of 305 high-resolution structures were selected to represent the set of known folds; 56 proteins were chosen that had at least one close structural match in this set. To test TOM, we attempted to determine which of the 305 folds was a match to each of the 56 protein sequences. TOM correctly predicts a close structural match for 45% of these proteins. THREADER, an algorithm chosen as a literature standard, correctly matched 20% of the test set. By comparing the performance of TOM, THREADER, and TOM NOVAR (a version of TOM without variability information), we conclude that the tendency of an amino acid to be buried or exposed is the dominant determinant of the success of threading algorithms. In addition, the structural alignments produced by TOM suggest that the exact alignment of just 30 to 50% of the residues in a sequence with the correct fold is necessary to select it as the highest scoring match in a set of folds.

Algorithms↗
PubMed1996

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

Amino Acid Sequence↗
PubMed1996

Separation of scrapie prion infectivity from PrP amyloid polymers.

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

1-Propanol↗
PubMed1996

A surface of minimum area metric for the structural comparison of proteins.

A new method for comparing protein structures, based on a minimal surface metric, is developed. A virtual polypeptide backbone is created by joining consecutive C alpha atoms in a protein structure. The minimal surface between the virtual backbones of two proteins (the Area Functional) is determined numerically using an iterative triangulation strategy. The first protein is then rotated and translated in space until the smallest minimal surface is obtained. Such a technique yields the optimal structural superposition between two protein segments. It requires no initial sequence alignment, is relatively insensitive to insertions and deletions, and obviates the need to select a gap penalty. The optimal minimal area can then be converted to the Area-C alpha distance, measured in angstroms, to determine the structural similarity. This technique has been applied to a large class of proteins and is able to detect not only small-scale differences between closely related proteins but also large-scale topological similarities between evolutionary unrelated proteins that lack any obvious sequence homology. To measure the similarity between structurally dissimilar proteins, an additional measure (the Fit Comparison) is developed. This is a scale-invariant measure of a structural similarity that is useful for determining topological similarities between dissimilar proteins with unrelated sequences.

Algorithms↗
PubMed1996

High-level expression and characterization of a purified 142-residue polypeptide of the prion protein.

The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.

Amino Acid Sequence↗
PubMed1996

An evolutionary trace method defines binding surfaces common to protein families.

X-ray or NMR structures of proteins are often derived without their ligands, and even when the structure of a full complex is available, the area of contact that is functionally and energetically significant may be a specialized subset of the geometric interface deduced from the spatial proximity between ligands. Thus, even after a structure is solved, it remains a major theoretical and experimental goal to localize protein functional interfaces and understand the role of their constituent residues. The evolutionary trace method is a systematic, transparent and novel predictive technique that identifies active sites and functional interfaces in proteins with known structure. It is based on the extraction of functionally important residues from sequence conservation patterns in homologous proteins, and on their mapping onto the protein surface to generate clusters identifying functional interfaces. The SH2 and SH3 modular signaling domains and the DNA binding domain of the nuclear hormone receptors provide tests for the accuracy and validity of our method. In each case, the evolutionary trace delineates the functional epitope and identifies residues critical to binding specificity. Based on mutational evolutionary analysis and on the structural homology of protein families, this simple and versatile approach should help focus site-directed mutagenesis studies of structure-function relationships in macromolecules, as well as studies of specificity in molecular recognition. More generally, it provides an evolutionary perspective for judging the functional or structural role of each residue in protein structure.

Amino Acid Sequence↗
PubMed1996

Secondary structure prediction and unrefined tertiary structure prediction for cyclin A, B, and D.

We present heuristic-based predictions of the secondary and tertiary structures of cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a beta-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure.

Amino Acid Sequence↗