Protein Stability, Folding and Design: Stabilization of scFv Protein through its reconstitution using a split GFB biosensor in vivo

General rules for rational design as well as prediction of tertiary structure and functionality of a protein can be described by investigating the interactions and the role of particular amino acids in protein structure. Mutagenesis has been used commonly to generate stable variants, with an ultimate goal to unravel the rules of protein stability and folding. Besides, reconstitution of dissected proteins has been used as well as an approach to find variants of particular proteins with increased affinity which could lead ultimately to enhancement of stability. In this project a random library of a hapten specific scFv, Anti Fluoroscien IsoThioCyanate (scFv) dissected into the fragments 1-124 (Heavy chain) and 125-246 (Light chain) was interrogated in order to find variants with improved affinity to be tested in further studies for stability enhancement of the corresponding intact protein variants. The split GFP system, a genetically codified biosensor, was used as a method to detect in vivo reconstitution of scFv (Heavy chain 1-124 + Light chain 125-246). Firstly, reconstitution of a single chain antibody (scFv) fragment 1-124 (Heavy chain) and 125-246 (Light chain) was detected. Secondly, a random library of the Light chain fragment 125-246 cloned into the GFP system was screened to find variants with higher fluorescence intensity than WT Light chain. An increase in fluorescence is suggested to arise from increased affinity which in turn could be used to select for stabilized intact variants. However we failed to detect green fluorescence. This may be due to problems in the expression of one of the partners (heavy chain-CGFP) or steric constraints and hence we were not able to screen any high affinity mutants. Various suggestions for improving the expression of the protein or relieving steric constraints are discussed here. If these problems are solved, libraries will be screened for the possible stabilizing role of the found substitutions. This can be discussed in terms of establishment of favorable hydrophobic interactions, stabilization of secondary structure and indirectly destabilization of the unfolded structure. The insight into the interactions and roles played by specific amino acids can be used to understand protein design of other proteins

Amyloid-β peptide 37, 38 and 40 individually and cooperatively inhibit amyloid-β 42 aggregation

The pathology of Alzheimer's disease is connected to the aggregation of β-amyloid (Aβ) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, Aβ37 and Aβ38, as pure peptides and in mixtures with Aβ40 and Aβ42. A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform Aβ42. Through kinetic analysis, we show that both Aβ37 and Aβ38 self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar β-sheet-rich aggregates, albeit on a longer timescale than Aβ40 or Aβ42. Additionally, we show that the shorter alloforms co-aggregate with Aβ40, affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither Aβ37 nor Aβ38 forms co-aggregates with Aβ42; however, both short alloforms reduce the rate of Aβ42 aggregation in a concentration-dependent manner. Finally, we show that the aggregation of Aβ42 is more significantly impeded by a combination of Aβ37, Aβ38, and Aβ40 than by any of these alloforms independently. These results demonstrate that the aggregation of any given Aβ alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain. This journal i

On the molecular mechanisms of the amyloid β-peptide aggregation

The pathogenesis of Alzheimer’s disease is widely believed to be due to production and deposition of the amyloid β-peptide. Several variants of the Aβ peptide are known to exist in in vivo. Variations include mutations or additional functional groups attached to residue side chains and may affect the aggregation process. Early on-set Alzheimer’s is caused by a variety of single amino acid substitutions of the Aβ peptide.The objectives of this thesis were to find a method to predict the aggregation propensity of Aβ40 variants and to understand the molecular mechanism of Aβ40 or Aβ42 peptide variants. We have extensively used thioflavin T-based fluorescence kinetic experiments to study the aggregation kinetics and the global analysis using the Amylofit platform to understand the molecular mechanism of aggregation of the variants. We showed that the aggregation propensity of Aβ40 variants can be predicted by monitoring the levels of inclusion body formation from peptides that are recombinantly expressed in E.coli cells. We could demonstrate that the net charge of a mutant greatly influences its aggregation propensity. We investigated the influence of various set of mutations on the aggregation mechanism of Aβ peptides. We showed that the aggregation behaviour is greatly modulated by the unstructured N-terminus of the Aβ peptide both in its 40- and 42-residue form. We could establish that specific residues at different positions in the primary sequence of Aβ40 peptide determined different stages of fibril formation. Especially, residues at positons 1, 7 and 13 of the Aβ40 peptide determined the specificity of secondary nucleation process of wild-type monomer. Besides this, we could confirm that the intact sequence of N-terminus is important in the aggregation process of Aβ42 peptide as reduced secondary nucleation rates were observed when residues at the N-terminus were scrambled. We addressed the influence of phosphorylation of two serine residues in the Aβ42 peptide on the aggregation process by designing single amino acid phosphomimic mutants. The rate of secondary nucleation is reduced more significantly for Ser26 substitutions than for Ser8 substitutions. Additionally, we found that Ser26 is a critical residue in the secondary nucleation of the Aβ42 peptide

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation.

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation

Amyloid-β peptide 37, 38 and 40 individually and cooperatively inhibit amyloid-β 42 aggregation.

The pathology of Alzheimer's disease is connected to the aggregation of β-amyloid (Aβ) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, Aβ37 and Aβ38, as pure peptides and in mixtures with Aβ40 and Aβ42. A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform Aβ42. Through kinetic analysis, we show that both Aβ37 and Aβ38 self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar β-sheet-rich aggregates, albeit on a longer timescale than Aβ40 or Aβ42. Additionally, we show that the shorter alloforms co-aggregate with Aβ40, affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither Aβ37 nor Aβ38 forms co-aggregates with Aβ42; however, both short alloforms reduce the rate of Aβ42 aggregation in a concentration-dependent manner. Finally, we show that the aggregation of Aβ42 is more significantly impeded by a combination of Aβ37, Aβ38, and Aβ40 than by any of these alloforms independently. These results demonstrate that the aggregation of any given Aβ alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain

The role of fibril structure and surface hydrophobicity in secondary nucleation of amyloid fibrils

Crystals, nanoparticles, and fibrils catalyze the generation of new aggregates on their surface from the same type of monomeric building blocks as the parent assemblies. This secondary nucleation process can be many orders of magnitude faster than primary nucleation. In the case of amyloid fibrils associated with Alzheimer's disease, this process leads to the multiplication and propagation of aggregates, whereby short-lived oligomeric intermediates cause neurotoxicity. Understanding the catalytic activity is a fundamental goal in elucidating the molecular mechanisms of Alzheimer's and associated diseases. Here we explore the role of fibril structure and hydrophobicity by asking whether the V18, A21, V40, and A42 side chains which are exposed on the Aβ42 fibril surface as continuous hydrophobic patches play a role in secondary nucleation. Single, double, and quadruple serine substitutions were made. Kinetic analyses of aggregation data at multiple monomer concentrations reveal that all seven mutants retain the dominance of secondary nucleation as the main mechanism of fibril proliferation. This finding highlights the generality of secondary nucleation and its independence of the detailed molecular structure. Cryo-electron micrographs reveal that the V18S substitution causes fibrils to adopt a distinct morphology with longer twist distance than variants lacking this substitution. Self- and cross-seeding data show that surface catalysis is only efficient between peptides of identical morphology, indicating a templating role of secondary nucleation with structural conversion at the fibril surface. Our findings thus provide clear evidence that the propagation of amyloid fibril strains is possible even in systems dominated by secondary nucleation rather than fragmentation

Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.

<p>Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.</p

Anti secretagogin Western blot analysis of endogenous secretagogin in BRIN-BD11 insulinoma cell lysates at selected concentrations of DTT.

<p><b>(5A)</b> Non-reducing Western blot of native secretagogin exposed for 30 s with low sensitivity Pierce^™ chemiluminescent Western blotting substrate (Thermo Scientific, cat. no. 32106). (5B) Non-reducing Western blot of native secretagogin exposed for 5 min with high sensitivity BM chemiluminescent Western blotting substrate (Roche, cat. no. 11500708001). Each lane has 30 μg of total protein with 20 mM DTT, 10 mM DTT, 5 mM DTT, 1 mM DTT, and no DTT respectively. M = monomer; D = dimer.</p

SDS-PAGE analysis of secretagogin dimers.

<p>(3A) Non-reducing gel of apo secretagogin. (3B) Non-reducing gel of calcium-loaded secretagogin. Lane 1 has pre stained protein ladder. Lanes 2–12 have 1 mg/mL secretagogin (30 μM) with 20 mM DTT, 10 mM DTT, 7 mM DTT, 5 mM DTT, 4 mM DTT, 3 mM DTT, 2 mM DTT, 1 mM DTT, 0.7 mM DTT (not present in 3B), 0.5 mM DTT and no DTT respectively. M = monomer; D = dimer; T = trimer (3C) Percent of monomer versus concentrations of DTT and dimer versus DTT concentration. Red: apo protein; blue: calcium-loaded protein; filled box: monomer; non-filled box: dimer.</p

The reversibility ranges of secretagogin analyzed by circular dichroism spectroscopy.

<p>Curves here represent temperature denaturation of both apo and calcium-loaded secretagogin during heating from 20–55°C and 20–65°C and from the maximum temperature to 20°C, for apo and calcium-loaded secretagogin at 0, 1 and 4 M urea and 0, 1 and 4 mM DTT. [Θ]_{222 nm} is ellipticity at 222 nm. Blue and pale blue dots represent calcium-loaded secretagogin during heating from 20°C to test temperature and reverse scan to 20°C, respectively. Red and pale red dots represent apo secretagogin during heating from 20°C to test temperature and reverse scan to 20°C, respectively. (A) [Θ]_{222 nm} versus temperature and total absorbance (nm) versus temperature at 20–55°C. (B) [Θ]_{222 nm} versus temperature and total absorbance (nm) versus temperature obtained at 20–65°C.</p