Data regarding the sensibility to proteolysis of a natural apolipoprotein A-I mutant.

The article shows dataset of the proteolysis of a natural variant of apolipoprotein A-I (apoA-I) with a substitution of a leucine by and arginine in position 60 (L60R), in comparison with the protein with the native sequence (Wt). This information demonstrates the potential of in vitro partial proteolysis experiments as it may be applicable to different approaches in the biophysical field. We have analyzed by different electrophoresis techniques apoA-I variants, quantified the degree of proteolysis after staining and compared the proteolysis efficiency with the computed cleavage patterns. The data shown here clearly strengthen the usefulness of this approach to test protein flexibility, as it may be attained with enzymes which are not expected to modify in vivo this protein but have a well-known digestion pattern. In addition it is appropriate for evaluating protein catabolism, as it is exemplified here by the evidence with metalloproteinase 12 (MMP-12), which is a physiological protease that may elicit the pro-inflammatory processing of this variant within the lesions. We support the work “Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis” (Gaddi, et al., 2020), gaining insights on protein folding from a characterization by proteolysis analysis [1].

© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license.

Value of the data
• Data shows a powerful design to compare arrangement, conformation and flexibility of proteins as it offers an inexpensive tool to support biophysical approaches. • These data may benefit to the extended field of protein studies as it may give information either on structure or on physiological catabolism. • This approach may be combined with mass spectrometry and bioinformatics analysis, and adapted to different proteins as a function of the information that is available on molecular weight or sequence.

Data description
Wt and L60R were incubated at increasing times with trypsin and reactions stopped after different time periods (0, 15, 30, 45 and 60 min). The digestion products were analyzed by  gradient gel electrophoresis (SDS-PAGGE) and developed by western blot using a specific antibody against apoA-I ( Fig. 1 ).
In order to determine whether the mutation may modify the digestion pattern, the computed mass of the peptides that could yield the proteolysis of the first 100 amino acids of apoA-I was analyzed ( Table 1 ). If complete, trypsin treatment should result in 14 peptides from 2 to 17 amino acid length. The replacement of the L by an R in the variant is predicted to split the dipeptide 60-61 into two single amino acids, thus incorporating a cleavage site.
Partial proteolysis mediated by chymotrypsin was also analyzed in the same trend. Together with trypsin, this enzyme is a potent tool for structural studies involving mass spectrometry and cross-linking [2] , as the cleavage pattern is well-defined (it selectively hydrolyzes peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine) [3] . Partial digestion of Wt and L60R is shown in Fig. 2 . Raw data of the performed analysis is shown as Supl. File 1 Reactions were stopped after 0, 15, 30, 45 and 60 min by addition of buffer containing 0.1% SDS following by 2-min boiling. Samples developed by the silver stain were loaded and resolved through SDS-PAGE. On the left, bands corresponding to the migration of the carbonic anhydrase (30 kDa) and trypsin inhibitor (20 kDa) were labeled to use as reference. B) Efficiency of the proteolysis was estimated by quantifying intensity of the band remaining within the original molecular weight (28 kDa) and normalized to the band intensity at time = 0 for each protein.
Circles and triangles correspond to Wt and L60R respectively ( * represents p < 0.05 with respect to the same time in the Wt) by using Student's test of triplicates. Again, we analyzed the peptides computed if complete proteolysis occurs of Wt (1-100) and L60R (1-100) ( Table 2 ). Interestingly, and in spite of the higher susceptibility, the addition of R (by substitution of L) removed the recognition site which cleaves the hexapeptide 58-64 into 58-60 and 61-64 in the Wt.
Finally, apoA-I proteolysis mediated by metalloproteinase-12 (MMP-12) was tested and the efficiency estimated by SDS-PAGE ( Fig. 3 ). Raw data for this analysis is also included in Supl. File 1 As a difference with the previous enzymes (which are not expected to participate in physiological roles involving apoA-I catabolism), MMP-12 is highly activated in leukocytes [4] , and seems to play, among other functions a detrimental role eliciting atherosclerotic lesion growth and increasing the susceptibility to rupture [5] . The existence of dysfunctional apoA-I in atherosclerotic plaques may suggest that it could be substrate of this or other pro inflammatory enzymes [6] . Unluckily, the proteolysis pattern is not possible to estimate as, even though it is proposed to cleave after alanine, valine, leucine, isoleucine, serine and threonine, the efficiency may vary on different peptides lengths [7] .

Protein variants purification
A Wt apoA-I cDNA template inserted into a pET-30 plasmid (Novagen, Madison, WI), was used as a template to obtain protein fused to a His-Tag peptide directly upstream of the Nterminus as previously described [8] . By using this template, the pro-amyloid mutant L60R was generated by the Quickchange method (Stratagene, La Jolla, CA) [9] [10] . This setup allowed protein isolation from E. coli lisates by nickel affinity columns. The His-Tag was further removed by chemical cleavage [11] , followed by a second metal affinity chromatography step to separate the final pure protein fraction. If required, proteins may be dialyzed against Tris 20 mM pH 7.4 (Tris buffer) and eluted through the same steps along the affinity column to obtain high quality of pure protein ( Fig. 4 ).

Partial degradation by proteolysis
In order to compare the influence of the substitution of a L by a R on the protein conformation, different enzymes were tested under conditions (molar ratios and time incubations), where proteolysis was not complete, thus allowing a comparison with the Wt. Variants were diluted to 0.3 mg/mL in Tris buffer, and incubated at 37 °C with mild agitation in the presence of trypsin, chymotrypsin or MMP-12, at molar ratios apoA-I variants to enzyme of 10 0 0:1, 50 0 0:1 and 500:1 respectively. After 0, 15, 30, 45 and 60 min, samples were combined with an appropriate amount of running buffer (containing 0.1% SDS) and heated in boiling water for 2 min. Following, variants incubated with trypsin were resolved by a gradient gel electrophoresis (12-24%) with SDS (SDS-PAGGE), and developed by western blotting using a polyclonal antibody against apoA-I [8] . Chymotripsin and MMP-12-treated samples were resolved by 16% SDS-PAGE, and developed by silver staining. The associated intensity of the remaining protein within the monomer molecular weight was quantified with the Image J 1.51 j8 software. Statistical differences were determined by the Student ś test analysis of triplicates of the samples treated under identical conditions in at least three different experiments. The relative intensity of L60R bands were compared with the same incubation time of Wt and normalized to the band intensity at time = 0. Significance is shown in each figure.

Other analytical methods
To predict whether the proteolysis pattern could be modified by the substitution of a L by a R, the software Peptide Mass, available through the ExPASy World Wide Web server [12] was run through the first 100 residues of Wt and L60R to compute the masses of the generated peptides following trypsin or chymotripsin treatment. No missed cleavage or post-translational modifications were allowed.
Protein content was quantified by the Bradford technique [13] or by absorbance from the estimation of the extinction coefficient (32,430 M −1 cm −1 at 280 nm) as determined in a Bio-Rad spectrophotometer (Hercules, CA).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Ethics statement
No animal or human samples have been used in this work.