04 WP Structure Forming Enzymes Jo Handouts

8/14/2019 04 WP Structure Forming Enzymes Jo Handouts

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PhD Course November 3 7, 2008

Whey proteins and structure forming proteases

1 Jeanette Otte - November 2008

Jeanette OtteFood Chemistry section

[email protected]

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


.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


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


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


Denaturation: temperature dependent (Fig. 9.15) Conformations:

Native Pre-MG Molten Globule Unfolded

(Fox & NcSweeney, 1998)

3D structure of -La

-lobe


-lobe

The Ca-binding cleft in -La

10 Jeanette Otte - November 2008 (Fox & McSweeney, 1998)

-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


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


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


A Gel

A visco-elastic solid composed of a smallamount of polymer (protein)forming a continous (crosslinked) network(the solid phase),

Aggregate


- 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, ...)


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


+ 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


pH optimum: high

1. Protease-induced gelation of WPI and -Lg



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Protease-induced gelation of WP


(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


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


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


(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


Mainly by non-covalent interactions

1 m


. - -


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6.000 x resolution:Needles and pins

Protease induced gelation of -lactalbumin:Microstructure (1)


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?




(Ipsen et al, 2000)

145.000 x resolution:-lactalbumin frag-

ments self-assemblesinto tubular struc-tureswhen BLP is added.




(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

?


1 Ca ++

Nanotubules20 nm diameterLong, stiff, brittle

Precipitate/AggregatesSupernatant

0 min

BLP-induced aggregates of -La

RP-HPLC

Hydrophilicpeptides Hydrophobic

-La


=> 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


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

31 Jeanette Otte - November 2008 31

Identification of -La fragments in aggregatesLC-MS

1 2 5 9 1 0 0 %

1 1 5 7 5 2 4 %

3 % -La


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


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

34 Jeanette Otte - November 2008 34= 11.259

3D structure of -La

35 Jeanette Otte - November 2008(Chrysina et al. 2000)

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


could explain aggregation with similar fragments?


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3. Formation of amyloid-like fibrils from -La


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


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


1

2

3

1

2

3

BLP-induced aggregates from 1% -Lacryo-TEM


Identification of -La fragments in aggregatesat 1% -LA

2 8 8 1 8

1 % -La

Rt 10.5 min


Retention time (min)

30 32 34 36 38 40 42 44 46

9 8

1 0 2 7 6


8/8

8

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


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


3D structure of -La

Formation of the8.8 kDa fragment


1-25+114-123

Mechanism of nanotubule and fibril formation


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


-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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04 WP Structure Forming Enzymes Jo Handouts