Kinetic analysis of amyloid protofibril dissociation and volumetric properties of the transition state

We present here the first detailed kinetic analysis of the dissociation reaction of amyloid protofibrils by utilizing pressure as an accelerator of the reaction. The experiment is carried out on an excessively diluted typical protofibril solution formed from an intrinsically denatured disulfide-deficient variant of hen lysozyme with Trp fluorescence as the reporter in the pressure range 3-400 MPa. From the analysis of the time-dependent fluorescence decay and the length distribution of the protofibrils measured on atomic force microscopy, we conclude that the protofibril grows or decays by attachment or detachment of a monomer at one end of the protofibril with a monomer dissociation rate independent of the length of the fibril. Furthermore, we find that the dissociation reaction is strongly dependent on pressure, characterized with a negative activation volume DeltaV(odouble dagger) = -50.5 +/- 1.60 ml mol(-1) at 0.1 MPa and with a negative activation compressibility Deltakappa(double dagger) = -0.013 +/- 0.001 ml mol(-1) bar(-1) or -0.9 x 10(-6) ml g(-1) bar(-1). These results indicate that the protofibril is a highly compressible high-volume state, but that it becomes less compressible and less voluminous in the transition state, most probably due to partial hydration of the existing voids. The system eventually reaches the lowest-volume state with full hydration of the monomer in the dissociated state.     

The Off-rate of Monomers Dissociating from Amyloid-β Protofibrils

The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.     

Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.     

Physiochemical characterization of the Alzheimer's disease-related peptides A beta 1-42Arctic and A beta 1-42wt

The amyloid beta peptide (A beta) is crucial for the pathogenesis of Alzheimer's disease. Aggregation of monomeric A beta into insoluble amyloid fibrils proceeds through several soluble A beta intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimer's disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimer's disease, and synthetic A beta 1-40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887-893]. To increase our understanding of the mechanisms involved in A beta aggregation, especially A beta monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of A beta 1-42wt and A beta 1-42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both A beta 1-42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes - monomer oligomerization and protofibril fibrillization - were affected differently by changes in the micro-environment and that the Arctic mutation alters the peptide response to such changes.     

Amyloid protofibril is highly voluminous and compressible

We report here results of the first direct measurement of partial volume and compressibility changes of a protein as it forms an amyloid protofibril. We use a high precision density meter and an ultrasonic velocity meter on a solution of intrinsically denatured, disulfide-deficient variant of hen lysozyme, and follow the time-dependent changes in volume and compressibility, as the protein spontaneously forms a protofibril. We have found a large increase in partial specific volume with time from 0.684 to 0.724 mL x g-1 (Deltanu = 0.040 mL x g-1 corresponding to 570 mL x (mol monomer)-1) and in partial specific adiabatic compressibility coefficient from -7.48 x 10(-12) to +1.35 x 10(-12) cm2 x dyn-1 (Deltabetas = 8.83 x 10(-12) x cm2 x dyn-1) as the monomer transforms into a protofibril. The results demonstrate that the protofibril is a highly voluminous and compressible entity, disclosing a cavity-rich, fluctuating nature for the amyloid protofibril. The volume and compressibility changes occur in two phases, the faster one preceding the major development of the beta-structure in the protofibril as monitored by CD.     

Conformation‐specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.     

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson's disease?

Parkinson's disease (PD) is one of many neurodegenerative diseases that are characterized by amyloid fibril formation. Alpha-synuclein is a primary component of the fibrillar neuronal inclusions, known as Lewy bodies, that are diagnostic of PD. In addition, the alpha-synuclein gene is linked to familial PD. Fibril formation by alpha-synuclein proceeds via discrete beta-sheet-rich oligomers, or protofibrils, that are consumed as fibrils grow. Both FPD mutations accelerate formation of protofibrils, suggesting that these intermediates, rather than the fibril product, trigger neuronal loss. In idiopathic PD, other factors may be responsible for accelerating protofibril formation by wild-type alpha-synuclein. One possible factor could be molecular crowding in the neuronal cytoplasm. We demonstrate here that crowding using inert polymers significantly reduced the lag time for protofibril formation and the conversion of the protofibril to the fibril, but did not affect the morphology of either species. Physiologically realistic changes in the degree of in vitro crowding have significant kinetic consequences. Thus, nonspecific changes in the total cytoplasmic protein concentration, induced by cell volume changes and/or altered protein degradation, could promote formation of and stabilize the alpha-synuclein protofibril.     

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.     

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion

Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer''s disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.     

Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.     

Abeta42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete Abeta42 species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.     

Pressure-accelerated dissociation of amyloid fibrils in wild-type hen lysozyme

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.     

Assembly of fibrin. A light scattering study.

Using stopped flow light scattering, we show that assembly of fibrin following activation with non-rate-limiting amounts of thrombin or reptilase occurs in two steps, of which the first is end-to-end polymerization of fibrin monomers to protofibrils and the second is lateral association of protofibrils to fibers, in agreement with Ferry's original proposal. Polymerization is found to proceed as a bimolecular association of bifunctional monomers; the overall rate varies as the inverse first power of the concentration; end-to-end association of two monomers, of a monomer and an oligomer, and of two oligomers occurs with the same rate constant. The value of the rate constant is 8.2 C 10(5) M-1 s-1 in 0.5 M NaCl, is three times larger in 0.1 M NaCl (0.05 M Tris, pH 7.4), and is the same following activation by reptilase and by thrombin. The onset of growth of fibers from protofibrils takes 12 times longer in 0.5 than in 0.1 M salt, i.e. thick fibers ("coarse" gels) form from short protofibrils, and thin fibers ("fine" gels) form from longer protofibrils. Jumps of salt concentration at times when protofibrils, but not fibers, have formed result in immediate growth of thick fibers at low salt from long protofibrils formed at high salt. The rate of fiber growth in these experiments varies as the inverse first power of the concentration. 3the instant of gelation (formation of a network of fibers) falls in the later half of the time during which the scattering rises due to fiber growth; the rise of gel rigidity after gelation is found to continue beyond the end of this period. Jumps from low to high salt result in retention of whatever fibers have formed at low salt and a very small additional increase of the scattering due to further fiber growth at high salt. From a variety of evidence, we conclude that the properties of fibrin are determined by kinetics and not equilibria of assembly steps. Results obtained here agree with the following scheme of fibrin assembly: monomers polymerize to protofibrils; long protofibrils associate laterally to fibers; occasionally a long protofibril associates with two different fibers to form an interfiber connection; fiber growth does not reverse to yield stabler, more compact, structures and terminates in formation of a network of fibers. The typical delay of fiber growth is the time during which protofibrils form from monomers. Measurements at rate-limiting concentrations of thrombin have allowed estimation of turnover rates of fibrinopeptides that agree with kinetic parameters obtained with direct assay of fibrinopeptide. Release of fibrinopeptide B causes more rapid fiber formation. Addition of thrombin after activation by reptilase, at a time when protofibrils, but not fibers, have formed, is followed rapidly by fiber formation; this proves that thrombin readily removes fibrinopeptide B from protofibrils. On the basis of these new results and earlier work (in particular, Blomb?ck, B., Hessel, B., Hogg, D., and Therkildsen, L...     

Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.