RICERCA ITALIANA     

Research program

Identification of folding and misfolding determinants by site-directed mutagenesis.

University Co-ordinator

Università degli Studi di PAVIA - BIOCHIMICA - ()

Research Unit Leader

Monica Stoppini

Description

The activity of our RU is focused on four principal topics: 1) production of b2-m variants, 2) determination of binding properties of b2-m mutants to collagen, 3) characterization of the kinetic parameters of amyloid fibrils formation by using three different fibrillogenesis assays, 4) optimization of the procedure of b2-m fibrillogenesis in conditions mimicking the biological micro-environment.

1) Beta2-m variants production.
We plan to generate two groups of variants: the first including mutants coming from systematic mutagenesis, each having one residue every three substituted by an alanine; the second group of variants will be selected on the basis of structural data giving to each residue to be replaced an important role in the physiological and pathological folding. In the latter approach three types of residues will be considered remarkable sites of mutation:
A) amino acids placed in more accessible surface regions in the fully folded protein
B) amino acids placed in more accessible surface regions, in the absence of strands I and VIII, probably involved in the partial unfolding of b2-m
C) amino acids in the protein core that, according to the NMR data obtained by RU of Udine, are proved to change their configuration during the slow phase of refolding (i.e L23, I35, V37, L40).
Following these criteria, a quite omogeneous distribution of mutations will be obtained, although not so regular as in the case of systematic mutagenesis performed every three residues. All charged and polar residues will be mutated to alanine, while the hydrophobic residues will be mutated to serine. Indeed most mutations (18 out of 30) involve charged residues. According to this experimental design ten mutations will involve residues normally interacting with the heavy chain of MHCI. Anyway systematic mutagenesis will be performed in the regions where a regular substitution 1/3 is not accomplished on the basis of the rational designing. According to these criteria, 40 mutants will be prepared. 12 of them have been already prepared in our lab and efficiently expresed and purified.
Expression of b2-m wild type and variants will be carried out as previously reported (21, 13) by using the plasmid pHN1 as a template. Mutagensis will be performed by using the QuikChange site-directed mutagenesis kit supplied by Stratagene. The procedure utilizes a supercoiled double-stranded DNA vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation. In order to realize our project a set of primers containing one or two mutations will be prepared. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by PfuTurbo DNA polymerase. Incorporation of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Afterwards Dpn I endonuclease is used to digest the parental DNA template. This endonuclease is very effective with the vector that we are using.
All the isoforms will be expressed as inclusion bodies and refolded as previously described (21).
All the recombinant variants will be purified and checked by mass spectrometry. Our unit will provide other four RU with all mutants for the following studies: I) investigation of the protein three dimensional structure in solution and generation of b2-m/MHCI complexes (Udine RU), II) characterization of b2-m fibrillogenesis in the presence of fibrillar collagen (Genoa RU), III) investigation of the accessibility to the solvent and structural flexibility by mass spectrometry associated to hydrogen exchange and limited proteolysis, IV) determination of the thermodynamic and kinetics properties of b2-m folding/unfolding (Florence RU).

2) Determination of binding properties of b2-m mutants to collagen isoforms.
For each b2-m mutant the binding properties to collagen type I and type II both from bovine and human source will be determined. These experiments will be performed on a BIAcore instrument as previously described for a limited number of b2-m variants (10), using triple helix collagen immobilized on sensorchips, while new immobilized matrixes will be prepared for type II and human collagens. We are going to establish for all mutant the dynamic properties of binding (kon and koff) as well as the binding at the equilibrium (kd). Experiments will be carried out at two pH compatible with the physio-pathologic environment (pH 6.4 and pH 7.4). The procedure will be also carried out in the presence of heparin at concentrations identical to those used in the fibrillogenesis (see the optimisation of fibrillogenesis) in order to establish if this glycosaminoglycan can interfere with the dynamic and static aspects of binding.

3) Fibrillogenesis.
This RU will perform three methods of fibrillogenesis involving different levels of conformational changes required for the amyloid fibrils formation:
A) extensive protein denaturation at acidic pH;
B) extensive maintaining of the structure and enhancement of hydrogen bonds in the presence of 20% trifluoroethanol;
C) overall conservation of the native structure in an environment enriched with biological factors (collagen and heparin) typically represented in the target tissue of this type of amyloidosis.

A) According to the first method, the fibrils are formed by incubating b2-m at acidic pH (in 50 mM NaCitrate, 100 mM NaCl, pH 2.5) at 37°C in the presence of seeds of preformed fibrils, as reported by Naiki et al. (22).
B) In the second procedure, the protein is incubated at 37 °C in 50 mM Na phosphate buffer, 100 mM NaCl, pH 7.4, in the presence of 20% trifluoroethanol and pre-formed fibrillar seeds. The fibrillogenesis will be monitored by thioflavin T fluorescence and confirmed with Congo red binding and electron microscopy.
C) The third method implies the use of biological factors able to favor the fibrillogenesis and it will be performed in close collaboration with the RU of Genoa. We will develope our recent observation regarding the role of the fibrillar collagen in the formation of amyloid fibrils on the collagen fibre (20). The aggregation experiments will be perfomed incubating b2-m with type I and II fibrillar collagen in 50mM ammonium acetate buffer, pH 6.4 at 37 °C and AFM will be used to monitor fibril formation in solution.
Type I fibrillar collagen will be prepared as previously described (10, 20) while for the preparation of the type II collagen a pre-extraction of glycosaminoglycans in the presence of 4M GdnHCl and following tripsynation of insoluble fibrils will be required.

4) Optimization of the procedure to form b2-m amyloid fibrils in conditions mimicking the biological micro-environment.
This RU will be also particularly involved in the development of new aspects of the third method of fibrillogenesis above described, in which so far type I fibrillar bovin collagen was used. We plan to develop a new test in which type II collagen, largely represented in the cartilage, and, above all, human collagen will be used. This collagen will be purified by samples of skin of normal subjects, undergoing the surgical removal of skin specimens (this study will be performed in accordance with the guidelines of the local ethical committees). Furthermore much attention will be dedicated to the possible role of heparin in combination with collagen in determining the fibril formation.
Preliminary data obtained by the RUs of Pavia and Genoa are encouraging because indicate that heparin, at a concentration varying from 1 to 10 ug/ml, significantly enhances the formation of the amyloid fibrils on the surface of collagen fibre. It is noteworthy that a therapeutic concentration of heparin around 10 ug/ml can be easily reached in plasma during the hemodialytic procedure.