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PhD Course November 3 7, 2008
Whey proteins and structure forming proteases
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Jeanette OtteFood Chemistry section
Whey proteins-Lg 3.5 g/L-La 1.2 g/L
Ig 0.7 g/L
High biological value
Interesting biological activity
Whey proteins
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.CMP ~ 1 g/L
LP ~ 30 mg/LLF 20-200 mg/LOsteopontinGrowth factorsEtc. etc.
Functional ingredients< Enzymatic modification,
e.g. proteolysis
-Lactoglobulin
162 aa, 18.3 kDa Compact, globular 3.6 x 3.6 nm
8 -strands forming a barrel binding small hydrophobic
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molecules, e.g. retinol Lipocalin family
2 S-S bonds 2 Trp and 4 Tyr
Genetic variants A, B,
(Fox & McSweeney, 1998; Holt & Sawyer, 2003)
-Lg in solution
Soluble in dilute salt pI 5.2 Association behaviour:
pH < 3.5 : monomer 18 kDa 3.5
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-Lactalbumin
123 aa, 14.2 kDa Compact, globular 2.3 x 2.6 x 4.0 nm
2 lobes: and
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Binds 1-2 mol Ca ++
4 S-S bonds 4 Trp and 4 Tyr
N-glycosylation (few %, Asn45)
Genetic variant B
(Brew, 2003)Asn45
-La in solution
Soluble in water, pI ~4.8 Metalloprotein: Apo-form : heat labile (55 C), renatures with
Ca 2+
Holo-form: more heat stable
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Denaturation: temperature dependent (Fig. 9.15) Conformations:
Native Pre-MG Molten Globule Unfolded
(Fox & NcSweeney, 1998)
3D structure of -La
-lobe
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-lobe
The Ca-binding cleft in -La
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-La: metal binding sites Primary Ca 2+ binding site:
In the loop, coordinated byAsp82, Asp87 and Asp88 carboxyl Oand Lys79 and Asp84 peptide carbonyl O
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Zn 2+ binding site:In the cleft, coordinated by Glu49 and Glu116
Second Ca 2+ binding site:Coordinated by Thr38, Glu39 and Asp83 sidechain O, and Leu81 peptide carbonyl O
(Brew, 2003)
Whey protein structures
Aggregates / polymers / particles A foam An emulsion An emulsion gel A gel Extruded roducts
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Microencapsulating agents Film and Fouling deposits
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OUTLINE
Protease-induced gelation of WPI and -Lg
Protease-induced gelation of -La
Protease-induced fibrils from -La
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A Gel
A visco-elastic solid composed of a smallamount of polymer (protein)forming a continous (crosslinked) network(the solid phase),
Aggregate
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- that immobilizes a large amountof water/solvent (the liquid phase)
ne- stranded
(Clark, 1992)
Gelation can be induced ...
Physically (heat , high pressure, ...)
Chemically (acid, alkali, salts, urea, alcohols,
changing pH, antibodies, ...)
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Enzymatically (cross-linking, proteolysis , .......)
Control
pH 3.0 pH 5.2 pH 7.0
Proteolysis affects microstructure andstrength of Heat-induced WPI Gels
1 m
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+ BLP
1 m*
(Otte et al. 1996a)
The enzymeB a c i l l u s l i c h e n i f o r m i s protease (BLP) from Novozymes A/S
A serine endoprotease specific for : Glu- and Asp-
Temperature optimum: 60C
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pH optimum: high
1. Protease-induced gelation of WPI and -Lg
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Protease-induced gelation of WP
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(Otte et al. 1996b)
Protease-induced gelation of -LgpH 7.5, 50C
- l a c
t o g
l o b u l
i n ( % )
60
70
80
90
100
n c e a t
5 0 0 n m
2
3
i c d i a m e
t e r
( n m
)
1000
1200
1400
1600
1800
2000
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Incubation time (min)
0 20 40 60 80 100 120 140
R e m a
i n i n g
-
20
30
40
50
A b s o r b a
0
1
H y d r o
d y n a m
0
200
400
600
800
(Otte et al. 1997)
1 m
Capillary electrophoresis of supernatant andprecipitate
Supernatant
12345
45
12345
0 min
10 min1 2 3 4 5 67
89
10 1112
13 14
-Lg
t 2 1 0 n m
Aggregates
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123
1234
5
Migration time (min)10 15 20 25 30 35
12345
70 min
100 min
130 min1 2 3 4 56
7 8 9
10 1112
13 14
A b s o r b a n c e
Migration time (min)10 15 20 25 30 35
1 7
6
9
1142
1
2 46
7
8
911 13
(Otte et al. 2000)
Primary peptide in -Lg aggregates
135 Lys-Phe-Asp-Lys-Ala-Leu-Lys-Ala-Leu-Pro- 145 Met-His-Ile-Arg-Leu-Ser-Phe-Asn-Pro-Thr- 155 Gln-Leu-Glu-Glu -
2825 Da
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(Otte et al. 2000)
QuestionAre there any structural features that could favour
aggregation of this peptide with the same or other peptides?
Hydrolysis of -Lg into 2.5 kDa peptidesFormation of Aggregates from most peptides
Primary peptide in aggregates is -Lg f135-157/158Aggregates associate into a Gel network
Protease-induced gelation of WPI and -LgM echanism
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Mainly by non-covalent interactions
1 m
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. - -
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6.000 x resolution:Needles and pins
Protease induced gelation of -lactalbumin:Microstructure (1)
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TEM image0 mM added Ca 2+ ,10% solutions, 2%BLP, pH 7.5, 50 oC
(Ipsen et al, 2000)
39.000 x resolution:Striking uniformity- but what are thosecircles?
Protease induced gelation of -lactalbumin:Microstructure (2)
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TEM image0 mM added Ca 2+ ,10% solutions, 2%BLP, pH 7.5, 50 oC
(Ipsen et al, 2000)
145.000 x resolution:-lactalbumin frag-
ments self-assemblesinto tubular struc-tureswhen BLP is added.
Protease induced gelation of -lactalbumin:Microstructure (3)
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TEM image0 mM added Ca 2+ ,10% solutions, 2%BLP, pH 7.5, 50 oC
(Ipsen et al, 2000)
How is -La transformed into tubules?
-LactalbuminCompact, globular,14.2 kDa, 4 nm123 aa, 4 S-S bonds8 Glu, 13 Asp
?
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1 Ca ++
Nanotubules20 nm diameterLong, stiff, brittle
Precipitate/AggregatesSupernatant
0 min
BLP-induced aggregates of -La
RP-HPLC
Hydrophilicpeptides Hydrophobic
-La
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=> Aggregates consist of hydrophobic peptides
R e t e n t i o n t i m e ( m i n )
2 0 3 0 4 0 5 0 60R e t e n t i o n t i m e ( m i n )
2 0 3 0 4 0 50 6 0
250 min
350 min
pept es
Hydrolysis and aggregation of -LaSize Exclusion Chromatography profiles
Rt 9.5 min Aggregates
Cryo-TEMand
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1
2
3
1
2
3-
1 = -La2 = large fragments3 = aggregates
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BLP-induced aggregates from 3% and 10% -Lacryo-TEM
Rt 9.5 min
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Identification of -La fragments in aggregatesLC-MS
1 2 5 9 1 0 0 %
1 1 5 7 5 2 4 %
3 % -La
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9 7 9 9
8 8 1 9
1 +
1 0 2 7
%
Rt 9.5 min
Identity of -La fragments
Measuredmass (Da)
-La f ragmen t
11,576 f12-113 cut once11,258 f12-46+50-113
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10,275 f12-37+50-11310,118 f26-113 cut once
9,800 f26-46+50-1138,817 f26-37+50-113
(Otte et al. 2005, Int. Dairy j. 15:219)
Primary sequence of -La
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3D structure of -La
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Questions
1. Which parts from the 3D structure of -La are cleaved off during formation of the 11.3 kDa fragment?
2. Does that result in exposure of any groups which
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could explain aggregation with similar fragments?
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3. Formation of amyloid-like fibrils from -La
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Amyloid fibrils
Associated with neurodegenerative diseases: Parkinsons disease,
Alzheimers disease, Type II diabetes, prion diseases (e.g.BSE), etc.
Bind Congo red and Thioflavine T
Formed by stacking of -sheets
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Typically 2-50 nm in diameter ?
Formation may involve: winding of protofilaments, protofibrils, fibrils,superfibrils
Unravelling the mechanism:Concentration determines structure
11.6 kg/mol
Calcium dependent
at least 3%
39 Jeanette Otte - November 2008 39Otte et al, 2004, submitted
8.8 kg/mol
Calcium independent
~1% and below
Hydrolysis and aggregation of -La at 1%SEC profiles
Rt 9.5 min Aggregates Rt 10.5 min
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1
2
3
1
2
3
BLP-induced aggregates from 1% -Lacryo-TEM
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Identification of -La fragments in aggregatesat 1% -LA
2 8 8 1 8
1 % -La
Rt 10.5 min
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Retention time (min)
30 32 34 36 38 40 42 44 46
9 8
1 0 2 7 6
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P e a
k a r e a a t
2 1 0 n m
0
5
10
15
20
25
30
-La11.576 Da fragment
8.818 Da fragment
v i n
f l u o r e s c e n c e
100
150 C
B
C
Thioflavinbinding
Formation of 8.8 kDa
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Reaction time (min)
0 50 100 150 200 250 300 350
M o
l a r
C D a
t 2 2 0 n m
-350
-300
-250
-200
T h i o f
l a
0
50
D
CD at 220 nm
Primary sequence of -La
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3D structure of -La
Formation of the8.8 kDa fragment
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1-25+114-123
Mechanism of nanotubule and fibril formation
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Conclusions
Gel structures can be formed by limitedproteolysis of WP (like CN) without heating
Different mechanisms for -Lg and -La
-Lg aggregates (~0.1 um) consist of manysmall peptides
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-La form dimeric aggregates of large fragments(~5 nm). These in turn assemble into tubules(~20 nm)
Alternatively, at low [Ca 2+ ] or [ -La], they formamyloid-like fibrils (5 nm)
The significance of these structures is not known
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