A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme.

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues, often with serious pathological consequences. Non-neuropathic systemic amyloidosis is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H.

Calculation of mutational free energy changes in transition states for protein folding.

Recent advances in experimental and computational methods have made it possible to determine with considerable accuracy the structures whose formation is rate limiting for the folding of some small proteins-the transition state ensemble, or TSE. We present a method to analyze and validate all-atom models of such structures. The method is based on the comparison of experimental data with the computation of the change in free energy of the TSE resulting from specific mutations.

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration.

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca.

Rapid formation of non-native contacts during the folding of HPr revealed by real-time photo-CIDNP NMR and stopped-flow fluorescence experiments.

We report the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, HPr, in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue. The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues, as (i) the observation of transient histidine photo-CIDNP signals during refolding of three of the mutants (F2W, F29W, and F48W) indicates a strong decrease in tryptophan accessibility to the flavin dye; (ii) iodide quenching experiments show that the quenching of the fluorescence of F48W is less efficient for the species formed during the dead-time of the stopped-flow experiment than for the fully native state; and (iii) kinetic fluorescence anisotropy measurements show that the tryptophan side-chain of F48W has lower mobility in the dead-time intermediate state than in both the fully denatured and fully native states. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues.

Protein folding and misfolding: a paradigm of self-assembly and regulation in complex biological systems.

Understanding biological complexity is one of the grand scientific challenges for the future. A living organism is a highly evolved system made up of a large number of interwoven molecular networks. These networks primarily involve proteins, the macromolecules that enable and control virtually every chemical process that takes place in the cell.

Characterization of single-tryptophan mutants of histidine-containing phosphocarrier protein: evidence for local rearrangements during folding from high concentrations of denaturant.

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching.

Structural dissection of alkaline-denatured pepsin.

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure.

Superficial granulomatous pyoderma treated with intravenous immunoglobulin.

Superficial granulomatous pyoderma is a rare variant of pyoderma gangrenosum. The superficial ulceration and vegetative margins are clinically distinctive; the suppurative and granulomatous histology is characteristic, though not pathognomonic. The condition is said to be relatively benign and more responsive to treatment than classic pyoderma gangrenosum, though published evidence of this is not always convincing.

In vitro characterization of lactoferrin aggregation and amyloid formation.

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.

Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril.

The molecular conformation of peptide fragment 105-115 of transthyretin, TTR(105-115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105-115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105-114.

De novo designed peptide-based amyloid fibrils.

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in beta-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric beta-sheets.

Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells.

Determination of a transition state at atomic resolution from protein engineering data.

We present a method for determining the structure of the transition state ensemble (TSE) of a protein by using phi values derived from protein engineering experiments as restraints in molecular dynamics simulations employing a realistic all-atom molecular mechanics energy function. The method uses a biasing potential to select an ensemble of structures having phi values in agreement with the experimental data set. An application to acylphosphatase (AcP), a protein for which phi values have been measured for 24 out of 98 residues, illustrates the approach.

The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation.

Amyloid fibrils and prions are proteinaceous aggregates that are based on a unique form of polypeptide configuration, termed cross-beta structure. Using a group of chemically distinct polyamino acids, we show here that the existence of such a structure does not require the presence of specific side chain interactions or sequence patterns. These observations firmly establish that amyloid formation and protein folding represent two fundamentally different ways of organizing polypeptides into ordered conformations.

Probing the exposure of tyrosine and tryptophan residues in partially folded proteins and folding intermediates by CIDNP pulse-labeling.

A nuclear magnetic resonance (NMR) technique has been devised to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues. Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulating rapid refolding of the protein. Crucial improvements in sensitivity were achieved by carrying out the polarization-producing photochemistry in a deoxygenated sample of the disordered state of the protein in a magnetic field of 4.

Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.

Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain.

Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS.

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form.

A kinetic study of beta-lactoglobulin amyloid fibril formation promoted by urea.

The formation of fibrillar aggregates by beta-lactoglobulin in the presence of urea has been monitored by using thioflavin T fluorescence and transmission electron microscopy (TEM). Large quantities of aggregated protein were formed by incubating beta-lactoglobulin in 3-5 M urea at 37 degrees C and pH 7.0 for 10-30 days.

Unfolding and aggregation during the thermal denaturation of streptokinase.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.

The protofilament structure of insulin amyloid fibrils.

Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-beta structure. However, there is a lack of information relating the 4.8 A beta-strand repeat to the higher order assembly of amyloid fibrils.

The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study.

The unfolding of the apo and holo forms of bovine alpha-lactalbumin (alpha-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein alpha-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and (1)H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with alpha-crystallin, have been obtained. alpha-LA contains four disulphide bonds and binds a calcium ion.