Protein folding Protein folding diseases Protein ... 02-14-03.pdf · Protein folding Protein...

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Proteins?

Protein function

Protein folding

Protein folding diseases

Protein interactions

Macromolecular assemblies

The end product of Genes

Protein Unfolding

N

H

C

R O

CC

O

N

C

DOD

D+

DOD

N

D

C

R O

CC

O

N

C

HDOD+

Acid Catalysis

OD-

N

H

C

R O

CC

O

N

C

DOD

N

D

C

R O

CC

O

N

C

HOD

N

H

C

R O

CC

O

N

C

DOD

D+

DOD

N

D

C

R O

CC

O

N

C

HDOD+

Acid Catalysis

Base Catalysis

0 2 4 6 8-4

-2

0

2

4

Log[

k(se

c-1)

]

pH

Amide Proton Exchange is pH dependent

EX1 mechanism: kch > kcl

Folded Open hydrogen exchangekcl

kop kch

N H N D

EX2 mechanism: kch < kcl

kex = kop

kex = K(op/cl) ·kch

“pH dependent”

Cytochrome C

Cooperative Unfolding

Englander, et. al. 2002

Cooperative Unfolding

Cytochrome CEnglander, et. al. 2002

Cytochrome C Folding Pathway

Hoang, et. al. 2002

Multi-state vs Two State Protein Unfolding

Englander, et. al. 2002

NMR H/D exchange

Individual amide proton exchange rates

Sensitive to subtle protein dynamics

Used extensively to study protein folding

Problems

Need pure sample

Need high concentrations

Only small proteins

Mass Spec H/D exchange?David Smith, (Zhang et al 1993)

Sample need not be pure

Low sample concentrations

Large proteins

Macromolecular complexes

Problems

Digestion coverage

Exchange rate is averaged over the whole peptide

Buffer intolerance

Mass spectrometry

Protein

Pepsin digestion (Optional)

•Exchange with D20•Quench with low pH over time

•Fragment size 10-20 residues

•Assignment of peptides•Quantifying exchange

H/D Exchange Experimental Protocol

0 2 4 6 8-4

-2

0

2

4

Log[

k(se

c-1)

]

pH

Protein stability“pulsed labeling H/D exchange”

[GdmCL]

0 1 2 3 4

0

20

40

60

80

100

Perce

ntage

excha

nge

[GdmCl]

MALDI Analysis of Pulse labeled proteins“SUPREX”

SUPREX; Stability of Unpurified Proteins from Rates of H/D Exchange

•Unpurified proteins

•Rapid analysis

•High throughput

•Protein stability in the cell

Ghaemmaghami, et. al. 2000

SUPREX

4-oxalocrotonate tautomerase

Suprex =

CD =

Powell, et. al. 2000

Maltose binding protein

SUPREX analysis of mutations and protein stability

Proteinstability

Ghaemmaghami, et. al. 2000

10 mM =56 mM =

Trp repressor (TrpR)- L-tryptophan =+ L-tryptophan =

Association detected by SUPREX

Powell, et. al. 2000

Protein Stability in Cells

Ghaemmaghami, et. al. 2001

MALDI Analysis of Pulse labeled proteins“SUPREX”

•Unpurified proteins

•Rapid analysis

•High throughput

•Protein stability in the cell

•?

Continous H/D Exchange

Protein folding

Protein interfaces

Quantitatively determine rates

?

Mass spectrometry

CA Tubes or Dimers

Pepsin digestion

•Exchange with D20•Quench with low pH over time

•Fragment size 10-20 residues

•Assignment of peptides•Quantifying exchange

H/D Exchange Experimental Protocol

Identification of protein interaction interfaces

DD

DD D

DD

D

DDH

H

HH H

HH

H

HH

Surface amideprotons

D2O

Protein Surface amide protonsreplaced withdeuterons

DD

DD D

DD

D

DD

HH

HH H

HH

H

HHH

H

HH D

DH

H

DH

H2O

Deuteriums trapped

Deuterium foot print

Protonated

Deuterated

Off exchange (10 min)

+ ATP

+ ATP & PKI

Detection of PKI Interaction with PKA

Mandell et al. 1998

PKI, PKA, and ATP

ATP

PKA

PKI

Mandell et al. 1998

Epitope mapping of a monoclonal antibodyagainst thrombin by H/D-exchange

mAB epitope

Novel Approaches for UnderstandingVirus Assembly and Dynamics.

Lanman et al. 2003

Image Reconstructions of Procapsid and Mature Virion

N-terminal domain21 kDa

C-terminal domain25 kDa

175 204

HINGE

The Coat Protein Subunit has a Two Domain Structure

Lanman, et. al. 1999

2

2

4

3

4

5

3

51

1

1 2 21

Monomer Mutant Dimer

Procapsid Expanded Lattice

N C

Unfolded Subunit

A domain-shuffling model for capsid expansion

The Model Predicts Trapping of Deuterium during Expansion

Transition State Expanded Procapsid

M ES XPS

M PS ES X

M PS XES M ES XPS M ES XPS

M ES XPS

M ES XPS

M ES XPS

M PS ES X

expanded shell protection

deuterium retention after expansion

procapsid shell protection

monomer protection

M ES XPS

M PS ES X

M PS XES M ES XPS M ES XPS

M ES XPS

M ES XPS

M ES XPS

MALNEGQIVT LAVDEIIETI SAITPMAQKA KKYTPPAASM QRSSNTIWMP VEQESPTQEG 60

WDLTDKATGL LELNVAVNMG EPDNDFFQLR ADDLRDETAY RRRIQSAARK LANNVELKVA 120

NMAAEMGSLV ITSPDAIGTN TADAWNFVAD AEEIMFSREL NRDMGTSYFF NPQDYKKAGY 180

DLTKRDIFGR IPEEAYRDGT IQRQVAGFDD VLRSPKLPVL TKSTATGITV SGAQSFKPVA 240

WQLDNDGNKV NVDNRFATVT LSATTGMKRG DKISFAGVKF LGQMAKNVLA QDATFSVVRV 300

VDGTHVEITP KPVALDDVSL SPEQRAYANV NTSLADAMAV NILNVKDART NVFWADDAIR 360

IVSQPIPANH ELFAGMKTTS FSIPDVGLNG IFATQGDIST L SGLCRIALW YGVNATRPEA 420

IGVGLPGQTA 430

M PS ES X

expanded shell protection

deuterium retention after expansion

procapsid shell protection

monomer protection

Changes in Exchange Protection during Assembly & Maturation

Tuma, et. al. 2000

TM

Immature Mature

SU

Gag

MA

CA

RNA

NC

MA CA p2 NC p1 p6

Gag

HIV Assembly & Maturation

Thin-Section EM Analysis of Assembly Products

CA is Comprised of Distinct N- and C- Terminal Structural Domains

N-domain

C-domain

CA Cylinders are Based on a Hexamer Lattice

From Li et al, Nature 407:409 (2000)

Hypothesis: The CA that does not form dimers, because of a mutation atthe dimer interface should form hexamers at high NaCl concentrations

+NaCl

+NaCl

CA does not form hexamers without C-domain (dimer) interaction.

Hypothesis: The CA that does not form dimers, because of a mutation atthe dimer interface should form hexamers at high NaCl concentrations

H/D exchange analyzed with FT-MS

Mass spectrometry

CA Tubes or Dimers

Pepsin digestion

•Exchange with D20•Quench with low pH over time

•Fragment size 10-20 residues

•Assignment of peptides•Quantifying exchange

H/D Exchange Experimental Protocol

2 3

56

1 4

Waste

Sample injection

C18 column

Pump AH2O, 0.05% Formic acid

Pump BACN, 0.05% Formic acid

ESI-MS Analysis of Peptides

MS

Sample loop

Ice bath (4 C)

Time

Ab

un

dan

ce

Elution of Digest from C4 Column

~1 min

m/z1 4001 2001 000 800 600

Representative FT-MS Scan

m/z 752 751 750 749 748 747 746 745

A

B

C

A = (744.842 * 2) Da

B = (746.390 * 2) Da

C = (749.386 * 1) Da

Expanded View of 744-752 m/z Region

744.842 within 1 ppm746.390 within 0.5 ppm

With High Resolution Peptides of Similar m/zcan be Analyzed

1PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG

!61GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTH

121NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE!!181VKNWMTETLL VQNANPDCKT ILKALGPGAT LEEMMTACQG VGGPGHKARV L

Peptides Covering 95% of the Sequence have Been Assigned

m/z 752 751 750 749 748 747 746 745

A

B

C

A = (744.842 * 2) Da

B = (746.390 * 2) Da

C = (749.386 * 1) Da

Expanded View of 744-752 m/z Region

m/z 752 751 750 749 748 747 746 745

40 mDa

Extremely High Resolution is Achievable with FT-MS

m/z 752 751 750 749 748 747 746 745

The Related Peaks are Analyzed toDetermine the Distribution

Isotopic distributions during H/D exchange

0 min

0.5 min

2 h

746 748 750m/z

0 min

0.5 min

2 h

Centroid = 745.5

Centroid = 748

Centroid = 749

746 748 750m/z

The Centroids of the Distribution are Calculated

Deuterium Incorporation at the Dimer Interface

68 h

0 sec

30 sec

2 min

1 h

4 h

849843 846m/z

H/D Exchange Period

Deuterium Incorporation Causes a Mass Increase

68 h

0 sec

30 sec

2 min

1 h

4 h

849843 846m/z

H/D Exchange Period

Assembled CA Unassembled CA

Deuterium Incorporation Causes a Mass Increase

68 h

0 sec

30 sec

2 min

1 h

4 h

849843 846m/z

H/D Exchange Period

Assembled CA Unassembled CA

Deuterium Incorporation Causes a Mass Increase

1 10 100 1000

1490

1491

1492

1493

1494

1495

1496

1497

1 10 100 10002530

2532

2534

2536

2538

2540

2542

1 10 100 1000

1524

1525

1526

1527

1528

1529

1530

1 10 100 1000

2136

2138

2140

2142

2144

2146

Time (min)

Hexamer1488 Monomer1488 F I Hex1488Sd mon1488Sd ### ### ### ###

Time (min)

Cen

troi

d of

Mas

s

hexamer2525 Monomer2525 mon2525fit Hex2525fit

Time (min)

Cen

troi

d of

mas

s

Hexamer1521 Monomer1521 Mon1521fit Hex1521fit

Hexamer2134 Monomer2134 Hex2134fit Hex2134fit3 Hex2134Sd

The Dimer Interface is Protected Upon Assembly

1 10 100 1000

1267

1268

1269

1270

1271

1272

Cen

troi

d of

Mas

s

Time (min)

Helix II Shows Protection Upon Assembly

Helices VI and VII Show Little Protection

1 10 100 10001933

1934

1935

1936

1937

1938

1939

1940Data: Dmean1932mon_MeanModel: 3expHD Chi^2 = 0.04466R^2 = 0.99146 A 1.45506 ±0.42438B 1.619 ±0.41511C 5.59556 ±0.32398k1 0.08275 ±0.04485k2 0.00548 ±0.00358k3 0.00015 ±0.00002

Cen

troi

d of

Mas

s

Time (min)

H1932mean M1932mean

1 10 100 1000

1490

1491

1492

1493

1494

1495

1496

1497

1498

Cen

troi

d of

Mas

s

Time (min)

The Bottom of Helix III becomes Protected

1 10 100 1000

1490

1491

1492

1493

1494

1495

1496

1497

1 10 100 10002530

2532

2534

2536

2538

2540

2542

1 10 100 1000

1524

1525

1526

1527

1528

1529

1530

1 10 100 1000

2136

2138

2140

2142

2144

2146

Time (min)

Hexamer1488 Monomer1488 F I Hex1488Sd mon1488Sd ### ### ### ###

Time (min)

Cen

troi

d of

Mas

s

hexamer2525 Monomer2525 mon2525fit Hex2525fit

Time (min)

Cen

troi

d of

mas

s

Hexamer1521 Monomer1521 Mon1521fit Hex1521fit

Hexamer2134 Monomer2134 Hex2134fit Hex2134fit3 Hex2134Sd

The Dimer Interface is Protected Upon Assembly

Helices VI and VII Show Little Protection

1 10 100 10001933

1934

1935

1936

1937

1938

1939

1940Data: Dmean1932mon_MeanModel: 3expHD Chi^2 = 0.04466R^2 = 0.99146 A 1.45506 ±0.42438B 1.619 ±0.41511C 5.59556 ±0.32398k1 0.08275 ±0.04485k2 0.00548 ±0.00358k3 0.00015 ±0.00002

Cen

troi

d of

Mas

s

Time (min)

H1932mean M1932mean

Changes in Exchange Rates due to CA Assembly

Lanman, et. al. 2003

SEC separation of cross-linked CA monomer and dimer

CAdst101901:jkl_UV1_280nm CAdst101901:jkl_UV1_280nm@01,COPY

0

100

200

300

400

mAU

0 20 40 60 80 100 ml

CA Monomer

CA Dimer

CA Monomer

CA Dimer

80 10020 40 600

Abso

rban

ce a

t 280

nm

Elution volume (ml)

Mass spectra of the cross-linked species

1000 1500 2000

0 10 20 30 40 50 60

0

50

100

150

200

250

10000 15000 20000

0 10 20 30 40 50 60

0

50

100

150

200

250

m/z

Ion

coun

t

Time (min)

m/z

Time (min)

10+11+

12+

13+

9+

8+

14439

Lys 182

Lys 70

Lysine 70 was cross-linked to Lysine 182

31 to 131 = 10948.9 171 to 199 = 3375.9 +DST = 114.0

Expected mass = 14438.8

Observed mass = 14439

Lys70 cross-linked to 182

12B

3A

A’

B’

An N-domain to C-domain Interaction in CA Assembly

C:C

N:C

N:N

Three Sites of Interaction During Assembly

Advantages of Hydrogen/Deuterium Exchange

•Small quantities required (10-12 mole)

•Needn’t be pure

•No symmetry constraints

•Can provide time resolved or dynamic information

The future of H/D exchangeMulti protein macromolecular complexes

Cooperativity in large proteins or macromolecular complexes

Exchange rates for individual amide protons

Protein dynamics during motor motions