Method for determining thermal stability of collagen or collagen-like peptide
View Patent ↗The present invention is a method for determining the thermal stability of a collagen peptide, collagen-like peptide or triple-helix construct with the repeating peptide unit Gly-Xaa 1 -Xaa 2 . The instant method accounts for the destabilizing effect of peptide repeats which do not conform to the highly stable Gly-Pro-Hyp peptide and for the interaction between triplets. The instant method finds use in mutant analysis of collagen peptides, collagen-like peptides or triple-helix constructs and engineering of collagen peptides, collagen-like peptides or triple-helix constructs.
1. A method for determining the thermal stability of a collagen peptide, collagen-like peptide or triple-helix construct comprising
a) obtaining the primary amino acid sequence of a collagen peptide, collagen-like peptide or triple-helix construct having consecutive repeating units of the tripeptide (Gly-Xaa 1 -Xaa 2 ) n , wherein Xaa 1 and Xaa 2 are any amino acid residue and n is at least 6;
b) identifying the number of consecutive Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct by computer inspection of the primary amino acid sequence of the collagen peptide, collagen-like peptide or triple-helix construct;
c) determining the maximum melting temperature of the collagen peptide, collagen-like peptide or triple-helix construct relative to the melting temperature of a (Gly-Pro-Hyp) n peptide, wherein n corresponds to the number of consecutive Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct;
d) identifying Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct wherein Xaa 1 is not Pro or Xaa 2 is not Hyp;
e) determining the melting temperature of each n repeat of step d) relative to the melting temperature of a Gly-Pro-Hyp peptide;
f) adjusting the melting temperature of each n repeat of step e) with a reference melting temperature for each n repeat of step e), wherein the reference melting temperature comprises:
Xaa 1 \Xaa 2
Hyp
Pro
Arg
Met
Ile
Gln
Pro
47.3
45.5
47.2
42.6
41.5
41.3
Glu
42.9
41.1
40.4
38.2
37.1
37.7
Ala
41.7
37.7
38.2
37.0
35.9
35.7
Lys
41.5
39.7
39.1
36.8
35.7
38.9
Arg
40.6
38.8
38.0
35.9
34.8
34.6
Gln
40.4
38.6
39.5
35.7
34.6
34.4
Asp
40.1
38.3
37.1
35.4
34.3
34.1
Leu
39.0
37.2
36.4
34.3
33.2
35.7
Val
38.9
37.1
36.3
34.2
33.1
32.9
Met
38.6
36.8
36.0
33.9
32.8
32.6
Ile
38.4
36.6
35.8
33.7
32.6
32.4
Asn
38.3
36.5
35.7
33.6
32.5
32.3
Ser
38.0
36.2
35.4
33.3
32.2
32.0
His
36.5
34.7
33.9
31.8
30.7
30.5
Thr
36.2
34.4
33.6
31.5
30.4
30.2
Cys
36.1
34.3
33.5
31.4
30.3
30.1
Tyr
34.3
32.5
31.7
29.6
28.5
28.3
Phe
33.5
31.7
30.9
28.8
27.7
27.5
Gly
33.2
31.4
30.6
28.5
27.4
27.2
Trp
31.9
30.1
29.3
27.2
26.1
25.9
Xaa 1 \Xaa 2
Ala
Val
Glu
Thr
Cys
Lys
Pro
40.9
40.0
39.7
39.7
37.7
36.8
Glu
34.6
35.3
35.3
35.9
33.3
35.0
Ala
32.9
34.4
34.1
34.1
32.1
30.8
Lys
35.1
34.2
35.3
33.9
31.9
31.0
Arg
34.2
33.3
33.8
33.0
31.0
29.5
Gln
34.0
33.1
32.8
32.8
30.8
32.6
Asp
31.6
32.8
32.5
32.5
30.5
30.9
Leu
31.2
31.7
31.4
31.4
29.4
31.1
Val
32.5
31.6
31.3
31.3
29.3
32.5
Met
32.2
31.3
31.0
31.0
29.0
31.7
Ile
33.9
31.1
30.8
30.8
28.8
27.9
Asn
31.9
31.0
30.7
30.7
28.7
27.8
Ser
31.6
30.7
30.4
30.4
28.4
27.5
His
30.1
29.2
28.9
28.9
26.9
26.0
Thr
29.8
28.9
28.6
28.6
26.6
25.7
Cys
29.7
28.8
28.5
28.5
26.5
25.6
Tyr
27.9
27.0
26.7
26.7
24.7
23.8
Phe
24.1
26.2
25.9
25.9
23.9
23.0
Gly
26.0
25.9
25.6
25.6
23.6
26.9
Trp
25.5
24.6
24.3
24.3
22.3
21.4
Xaa 1 /Xaa 2
His
Ser
Asp
Gly
Leu
Asn
Pro
35.7
35.0
34.0
32.9
32.7
31.7
Glu
31.3
30.6
29.7
28.5
28.3
29.5
Ala
30.1
33.0
33.0
27.3
27.8
26.1
Lys
29.9
29.2
35.8
27.1
26.9
31.7
Arg
29.0
30.5
34.5
26.2
26.0
25.0
Gln
28.8
28.1
27.1
26.0
25.8
24.8
Asp
28.5
27.8
26.8
25.7
25.5
24.5
Len
27.4
26.7
25.7
24.6
26.9
23.4
Val
27.3
26.6
25.6
24.5
24.3
23.3
Met
27.0
26.3
25.3
24.2
24.0
23.0
Ile
26.8
26.1
25.1
24.0
23.8
22.8
Asn
26.7
26.0
25.0
23.9
23.7
22.7
Ser
26.4
25.7
24.7
23.6
23.4
22.1
His
24.9
24.2
23.2
22.1
21.9
20.9
Thr
24.6
23.9
22.9
21.8
21.6
20.6
Cys
24.5
23.8
22.8
21.7
21.5
20.5
Tyr
22.7
22.0
21.0
19.9
19.7
18.7
Phe
21.9
21.2
20.2
19.1
18.9
17.9
Gly
21.6
20.9
19.9
18.8
25.3
17.6
Trp
20.3
19.6
18.6
17.5
17.3
16.3
Xaa 1 /Xaa 2
Tyr
Phe
Trp
Pro
30.2
28.4
26.1
Glu
25.8
24.0
21.7
Ala
24.6
21.9
20.5
Lys
24.4
22.6
20.3
Arg
23.5
21.7
19.4
Gln
23.3
21.5
19.2
Asp
23.0
21.2
18.9
Len
21.9
20.1
17.8
Val
21.8
20.0
17.7
Met
21.5
19.7
17.4
Ile
21.3
19.5
17.2
Asn
21.2
19.4
17.1
Ser
20.9
19.1
16.8
His
19.4
17.6
15.3
Thr
19.1
17.3
15.0
Cys
19.0
17.2
14.9
Tyr
17.2
15.4
13.1
Phe
16.4
14.6
12.3
Gly
16.1
19.7
12.0
Trp
14.8
13.0
10.7
g) combining the adjusted melting temperatures of the n repeats of steps f) so that a correction melting temperature is determined for Gly-Xaa 1 -Xaa 2 repeats; and
h) adjusting the maximum melting temperature of the collagen peptide, collagen-like peptide or triple-helix construct of step c) with the correction melting temperature of g) to determine the thermal stability of the collagen peptide, collagen-like peptide or triple-helix construct; and
i) identifying the presence of a stabilizing sequence motif in the collagen peptide, collagen-like peptide or triple-helix construct, wherein the stabilizing sequence motif comprises Lys-Gly-Glu/Asp, and adjusting the melting temperature of step h) for the collagen peptide, collagen-like peptide or triple-helix construct when the stabilizing sequence motif is present.
2. The method of claim 1 , wherein the collagen peptide, collagen-like peptide or triple-helix construct is within a protein or collagen domain of a protein.
3. The method of claim 2 , wherein a disease-causing mutation is present in the protein.
4. A method for designing a collagen peptide, collagen-like peptide or triple-helix construct with a predetermined thermal stability comprising
a) obtaining the primary amino acid sequence of a collagen peptide, collagen-like peptide or triple-helix construct having consecutive repeating units of the tripeptide (Gly-Xaa 1 -Xaa 2 ) n , wherein Xaa 1 and Xaa 2 are any amino acid residue and n is at least 6;
b) identifying the number of consecutive Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct by computer inspection of the primary amino acid sequence of the collagen peptide, collagen-like peptide or triple-helix construct;
c) determining the maximum melting temperature of the collagen peptide, collagen-like peptide or triple-helix construct relative to the melting temperature of a (Gly-Pro-Hyp) n peptide, wherein n corresponds to the number of consecutive Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct;
d) identifying Gly-Xaa 1 -Xaa 2 amino acid repeats in the collagen peptide, collagen-like peptide or triple-helix construct wherein Xaa 1 is not Pro or Xaa 2 is not Hyp;
e) determining the melting temperature of each n repeat of step d) relative to the melting temperature of a Gly-Pro-Hyp peptide;
f) adjusting the melting temperature of each n repeat of step e) with a reference melting temperature for each n repeat of step e), wherein the reference melting temperature comprises:
Xaa 1 \Xaa 2
Hyp
Pro
Arg
Met
Ile
Gln
Pro
47.3
45.5
47.2
42.6
41.5
41.3
Glu
42.9
41.1
40.4
38.2
37.1
37.7
Ala
41.7
37.7
38.2
37.0
35.9
35.7
Lys
41.5
39.7
39.1
36.8
35.7
38.9
Arg
40.6
38.8
38.0
35.9
34.8
34.6
Gln
40.4
38.6
39.5
35.7
34.6
34.4
Asp
40.1
38.3
37.1
35.4
34.3
34.1
Leu
39.0
37.2
36.4
34.3
33.2
35.7
Val
38.9
37.1
36.3
34.2
33.1
32.9
Met
38.6
36.8
36.0
33.9
32.8
32.6
Ile
38.4
36.6
35.8
33.7
32.6
32.4
Asn
38.3
36.5
35.7
33.6
32.5
32.3
Ser
38.0
36.2
35.4
33.3
32.2
32.0
His
36.5
34.7
33.9
31.8
30.7
30.5
Thr
36.2
34.4
33.6
31.5
30.4
30.2
Cys
36.1
34.3
33.5
31.4
30.3
30.1
Tyr
34.3
32.5
31.7
29.6
28.5
28.3
Phe
33.5
31.7
30.9
28.8
27.7
27.5
Gly
33.2
31.4
30.6
28.5
27.4
27.2
Trp
31.9
30.1
29.3
27.2
26.1
25.9
Xaa 1 \Xaa 2
Ala
Val
Glu
Thr
Cys
Lys
Pro
40.9
40.0
39.7
39.7
37.7
36.8
Glu
34.6
35.3
35.3
35.9
33.3
35.0
Ala
32.9
34.4
34.1
34.1
32.1
30.8
Lys
35.1
34.2
35.3
33.9
31.9
31.0
Arg
34.2
33.3
33.8
33.0
31.0
29.5
Gln
34.0
33.1
32.8
32.8
30.8
32.6
Asp
31.6
32.8
32.5
32.5
30.5
30.9
Leu
31.2
31.7
31.4
31.4
29.4
31.1
Val
32.5
31.6
31.3
31.3
29.3
32.5
Met
32.2
31.3
31.0
31.0
29.0
31.7
Ile
33.9
31.1
30.8
30.8
28.8
27.9
Asn
31.9
31.0
30.7
30.7
28.7
27.8
Ser
31.6
30.7
30.4
30.4
28.4
27.5
His
30.1
29.2
28.9
28.9
26.9
26.0
Thr
29.8
28.9
28.6
28.6
26.6
25.7
Cys
29.7
28.8
28.5
28.5
26.5
25.6
Tyr
27.9
27.0
26.7
26.7
24.7
23.8
Phe
24.1
26.2
25.9
25.9
23.9
23.0
Gly
26.0
25.9
25.6
25.6
23.6
26.9
Trp
25.5
24.6
24.3
24.3
22.3
21.4
Xaa 1 /Xaa 2
His
Ser
Asp
Gly
Leu
Asn
Pro
35.7
35.0
34.0
32.9
32.7
31.7
Glu
31.3
30.6
29.7
28.5
28.3
29.5
Ala
30.1
33.0
33.0
27.3
27.8
26.1
Lys
29.9
29.2
35.8
27.1
26.9
31.7
Arg
29.0
30.5
34.5
26.2
26.0
25.0
Gln
28.8
28.1
27.1
26.0
25.8
24.8
Asp
28.5
27.8
26.8
25.7
25.5
24.5
Len
27.4
26.7
25.7
24.6
26.9
23.4
Val
27.3
26.6
25.6
24.5
24.3
23.3
Met
27.0
26.3
25.3
24.2
24.0
23.0
Ile
26.8
26.1
25.1
24.0
23.8
22.8
Asn
26.7
26.0
25.0
23.9
23.7
22.7
Ser
26.4
25.7
24.7
23.6
23.4
22.1
His
24.9
24.2
23.2
22.1
21.9
20.9
Thr
24.6
23.9
22.9
21.8
21.6
20.6
Cys
24.5
23.8
22.8
21.7
21.5
20.5
Tyr
22.7
22.0
21.0
19.9
19.7
18.7
Phe
21.9
21.2
20.2
19.1
18.9
17.9
Gly
21.6
20.9
19.9
18.8
25.3
17.6
Trp
20.3
19.6
18.6
17.5
17.3
16.3
Xaa 1 /Xaa 2
Tyr
Phe
Trp
Pro
30.2
28.4
26.1
Glu
25.8
24.0
21.7
Ala
24.6
21.9
20.5
Lys
24.4
22.6
20.3
Arg
23.5
21.7
19.4
Gln
23.3
21.5
19.2
Asp
23.0
21.2
18.9
Len
21.9
20.1
17.8
Val
21.8
20.0
17.7
Met
21.5
19.7
17.4
Ile
21.3
19.5
17.2
Asn
21.2
19.4
17.1
Ser
20.9
19.1
16.8
His
19.4
17.6
15.3
Thr
19.1
17.3
15.0
Cys
19.0
17.2
14.9
Tyr
17.2
15.4
13.1
Phe
16.4
14.6
12.3
Gly
16.1
19.7
12.0
Trp
14.8
13.0
10.7
g) combining the adjusted melting temperatures of the n repeats of steps f) so that a correction melting temperature is determined for Gly-Xaa 1 -Xaa 2 repeats;
h) adjusting the maximum melting temperature of the collagen peptide, collagen-like peptide or triple-helix construct of step c) with the correction melting temperature of g);
i) identifying the presence of a stabilizing sequence motif in the collagen peptide, collagen-like peptide or triple-helix construct, wherein the stabilizing sequence motif comprises Lys-Gly-Glu/Asp, and adjusting the melting temperature of step h) for the collagen peptide, collagen-like peptide or triple-helix construct when the stabilizing sequence motif is present thereby determining the thermal stability of the collagen peptide, collagen-like peptide or triple-helix construct; and
j) modifying the primary amino acid sequence of the collagen peptide, collagen-like peptide or triple-helix construct to obtain a collagen peptide, collagen-like peptide or triple-helix construct with a predetermined thermal stability.