Jennifer Reed

Aggregation and secondary structure of synthetic amyloid βA4 peptides of Alzheimer's disease☆

The deposition of amyloid beta A4 in the brain is a major pathological hallmark of Alzheimers disease. Amyloid beta A4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural beta A4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of beta A4. A comparison of amino-terminally truncated beta A4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid beta A4. Infrared spectroscopy of beta A4 peptides in the solid state shows that their secondary structure consists of a beta-turn flanked by two strands of antiparallel beta-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative beta-turn positions. Limited proteolysis of these analogs allowed a localization of the central beta-turn at residues 26 to 29 of the entire sequence. Purified beta A4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a beta-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a beta-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the beta A4 sequence. This model is sustained by the properties of beta A4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between beta A4 peptides.

Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues.

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collision offset and of the charge state of the molecular ion on phosphate-specific fragmentation processes was investigated in detail. H--D exchange experiments and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C-beta hydrogen of homoserine is transferred to the protonated phosphate group. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these phosphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the negative ion product spectra, which is consistently elevated in tyrosine phosphopeptides as compared with serine, threonine and homoserine phosphopeptides. The fragment ions of methylphosphono- and H-phosphonopeptides can be explained by the same mechanisms as are applicable to phosphopeptides.

Direct Effects of Phosphorylation on the Preferred Backbone Conformation of Peptides: A Nuclear Magnetic Resonance Study

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Halpha coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

Phosphorylated human galectin-3: Facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy

Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.

Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species☆

Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.

Modelling of the human papillomavirus type 16 E5 protein

The product of the E5 oncogene in human papillomaviruses (HPVs) participates in cellular transformation. The sequences of E5 from high-risk HPV types are closely related, and the ability to transform is thought to be associated with their structure. Structural determination by standard biophysical methods has proved impossible due to the extreme hydrophobicity of the gene product. We have achieved limited solubility by dividing the sequence into three, structurally distinct domains. Synthetic peptides corresponding to these domains have been examined using circular dichroism (CD) spectroscopy, a method that can detect secondary structure elements in highly dilute protein solutions. Using data on the secondary structure content of these domains under different conditions and in systematic combination to detect constructive domain interactions, a model of HPV E5 structure and position in the membrane is proposed that is consistent with what is known of the larger family of leucine-rich repeat (LRR) proteins to which it belongs.

Influence of Chelate Conjugation on a Newly Identified Tumor-Targeting Peptide

The transfer of peptide sequences identified by screening of phage-displayed libraries to clinical application is often difficult. This study investigated whether coupling of a new peptide, FROP-1, to the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) resulted in structural restriction and, consequently, improved binding and stability. Methods: The peptide FROP-1 was coupled to the chelator DOTA and labeled with 111In. The structural changes caused by the addition of the chelator were determined by circular dichroism. The properties of this modified peptide were investigated in in vitro binding assays and monitored for kinetics, competition, and internalization as well as serum stability. A cell-type binding profile was established and the in vivo biodistribution was evaluated in a nude mouse model. Results: When compared with the free peptide without chelator, FROPDOTA revealed different cellular uptake kinetics, reaching a maximum at 2 h in vitro. The cells completely accumulated the tracer, and competition experiments revealed that 99.4% (FRO82-2 cells), 98.6% (MCF-7 cells), or 99.3% (average for 3 primary head and neck tumor cell lines) of tracer accumulation could be suppressed, revealing the specificity of this process. The internalization kinetics determined in MCF-7 cells supported this finding: After an incubation time of 180 min, the major fraction of FROPDOTA was trapped intracellularly. Serum stability experiments revealed an increase in stability due to the chelator, with a half-life of 71 min. Circular dichroism measurements indicated a fixed α-helix structure of FROPDOTA representing a strong change in secondary structure. In competition binding experiments, the binding constant (KD) to FRO82-2 cells was determined to be 494 nM. Despite this avid binding affinity, the binding kinetics were found to be too slow to induce an uptake in vivo before clearance. Consequently, the biodistribution revealed a rapid renal and hepatobiliary clearance, with blood levels dropping from 5.48 ± 0.26 %ID/g (percentage injected dose per gram) 5 min after injection to 0.77 ± 0.15 %ID/g at 135 min after injection. Conclusion: This study revealed that peptides that are identified by display techniques may be underrated. Careful alteration of their structure will permit going beyond the possibilities that the limited pool of naturally occurring peptides provide for tumor targeting.

Effect of Variation of the Strength of the Aromatic Interactions of Tryptophan on the Cooperative Structural Refolding Behavior of a Peptide from HIV 1

A 15-residue sequence (LPCRIKQFINMWQEV) forming the principal CD4-binding domain of gp120 from HIV 1 displays unusual, highly cooperative refolding from beta-hairpin to 3(10) helix when the polarity of the surrounding medium drops below a critical point, the so-called conformational switch. The tryptophan at position 12 has been shown to be essential for the cooperativity of the refolding process, and several lines of evidence from earlier work had suggested that it was the aromatic quadrupole that was responsible for this. To define more precisely what physico-chemical properties of tryptophan brought about the unique behavior of this peptide, nonproteogenic aromatic amino acids have been selected based on desired alterations in quadrupole moment, electrostatic potential surface, and binding energy to ions. These were built into the peptide in the place of tryptophan and their effect on switch behavior examined. It could be shown that a minimal strength of the quadrupole moment is necessary but not sufficient to enforce cooperativity of refolding, with other properties of tryptophan playing a role in the optimum interaction of this residue with other side chains of the peptide.

Isolation and elucidation of some functional properties of the “mute” catalytic subunit of cAMP-dependent protein kinase

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.

Transtactin: a universal transmembrane delivery system for Strep-tag II-fused cargos.

The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD‐fused Strep‐Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep‐tag II‐linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin‐introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep‐tag II‐fused cargos.