Structure of the α-Helix - UAB a Theoretical Lab ; 4 minutes . In a UAB Lab : 7 minutes . On Ice...

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Structure of the α-Helix

Transcript of Structure of the α-Helix - UAB a Theoretical Lab ; 4 minutes . In a UAB Lab : 7 minutes . On Ice...

Structure of the α-Helix

Structure of the β−Sheet

Protein Dynamics

Basics of Quenching HDX

Hydrogen exchange of amide protons is catalyzed by H2O, OH-

, and H3O+, but it’s most dominated by base catalyzed reactions.

Ka 6.95E-01 Kb 1.87E+08 Kw 5.27E-04

Place of Quenched Reaction

Half Life of Back Exchange

My hand 1 minute

In a Theoretical Lab 4 minutes

In a UAB Lab 7 minutes

On Ice (0C) 1 hour

In the -20C Fridge 14 hours

In the -80C Fridge 7 Years

In Liquid Nitrogen When Hell Freezes Over

In addition to lowering the pH, chemical kinetics are considerable slowed with decreasing temperature.

Kinetics of HDX For exchange to occur: 1. The site needs to be at least transiently exposed to the bulk solvent. 2. The site must be available to form new hydrogen bonds. If it is either exposed with an unavailable site for hydrogen bonding or buried (most always hydrogen bonded in this case), it is described as protected.

In HDX experiments, the degree of protection (Pf) is empirically determined by the extent the rate of exchange (kex) is slowed from the predicted chemical rate (kch) . 0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6

Deut

eriu

m U

ptak

e

Time (s)

Kch Kex

Kinetic Range of Protection

0

0.2

0.4

0.6

0.8

1

1.2

0 0.25 0.5 0.75 1

Unprotectedlog Pf 0.5

log Pf 1

log Pf 2

log Pf 3

log Pf 4

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60Deu

teriu

m U

ptak

e

Unprotectedlog Pf 0.5

log Pf 1

log Pf 2

log Pf 3

log Pf 4

0

0.2

0.4

0.6

0.8

1

1.2

0 200 400 600Time (s)

Unprotectedlog Pf 0.5

log Pf 1

log Pf 2

log Pf 3

log Pf 4

A wide span of different kinetics can be observed for the range of protection that can occur within a single protein.

Kinetic Range of Protection

0

0.2

0.4

0.6

0.8

1

1.2

0.001 0.1 10 1000

Deu

teriu

m U

ptak

e

Time (s)

Unprotectedlog Pf 0.5

log Pf 1

log Pf 2

log Pf 3

log Pf 4

Log Pf 6

A wide span of different kinetics can be observed for the range of protection that can occur within a single protein.

Because of this, the data is more reasonably visualized on the log scale.

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Sample Injection *

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Sample Injection *

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Online Pepsin Digestion

*

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation and Analysis

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation and Analysis

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation and Analysis

0°C

-

1°C

-1°C

Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Colum

n Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation and Analysis

0°C

-1°C

-1°C Sample Loop

Waste

Quaternary Pump

0°C

Pepsin Column

Binary Pump

Trap

Waste

C18

QTOF

Peptide Separation and Analysis *

Reproducibility and Peptide Diversity

HuTrim5a Coverage Map

FRACTION BURIED

0-0.2

0.2-0.4

0.4-0.6

Aspergillus Protease Complements Pepsin

• Calculate isotope distribution using known chemical composition

• Calculate theoretical spectra for peptide +1, +2, +3 …. Deutrons

• Fit experimental spectra with Gaussian distributed sum of calculated spectra

• Assess quality of fit, calculate percent exchange

Data Analysis using HDExaminer

0.50s

1s

3s

10s

12s

30s

90s

5m

15m

45m

2h

8h

0.50s

1s

3s

10s

12s

30s

90s

5m

15m

45m

2h

8h

0.50s

1s

3s

10s

12s

30s

90s

5m

15m

45m

2h

8h

121

181

Q S L R E L I S D L E H R L Q G S V M E L L Q G V D G V I K R T E N V T L K K P E T F P K N Q R R V F R A P D L K G M L

E V F R E L T D V R R Y W V D V T V A P N N I S C A V I S E D K R Q V S S P K P Q I I Y G A R G T R Y Q T F V N F N Y C

T G I L G S Q S I T S G K H Y W E V D V S K K T A W I L G V C A G F Q P D A M C N I E K N E N Y Q P K Y G Y W V I G L E 241

125 130 135 140 145 150 155 160 165 170 175 180

185 190 195 200 205 210 215 220 225 230 235 240

245 250 255 260 265 270 275 280 285 290 295 300

Heat Map Showing Exchange Kinetics

Effect of pH on Tail Needle

TM

Immature Mature

SU

Gag

MA

CA

RNA

NC

MA CA p2 NC p1 p6

Gag

HIV Assembly & Maturation

Immature and Mature Virions are Pleomorphic

Immature Mature

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

N-domain

C-domain

H1

Cyclophilin loop

H1

CA Cylinders are Based on a Hexamer Lattice

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

+NaCl

CA does not Form Hexamers without the C-Domain (dimer) Interaction

Mass spectrometry

CA Dimers and Tubes

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

Changes in Exchange Rates due to CA Assembly

H4

H9

H1

Crosslinking of Intact Tubes will Allow Identification of Intersubunit Interfaces

Lysine 70 was Cross-Linked to Lysine 182

31 to 131 = 10 948.9 171 to 199 = 3 375.9 +DST = 114.0 Expected mass = 14 438.8 Observed mass = 14 439

Lys70 cross-linked to 182 Lys 70

Lys 182

1 2

B

3 A

A’

B’

A Model for An N-domain to C-domain Interaction in CA Assembly

Immature viral-like particles

Mature viral-like particles

Hydrogen Deuterium Exchange Studies on Virus-Like Particles

CAfast is Partially Protected at Early Times

30 sec

5 min

45 min

CA in Mature Virus-Like Particles is 50% Free and 50% Core-Incorporated

+ CAslow CAfast

The N:C Interface is Bimodal in Mature Virions

m/z752750748746

746 748 750 752

m/z

0 sec

20 sec

4 min

16 min

1 h

4 h

16 h Mature virus- like particles

Immature virus- like particles

The N:C Interface is Not Formed in Immature Virions

m/z 0 sec

20 sec

4 min

16 min

1 h

4 h

16 h

m/z752750748746746 748 750 752

1 2 B 3

A

A’

B’

1 B

A 3 A’

B’

+

The N-domain:C-domain Interaction is Crucial for Viral Maturation

3

A

A’

C:C

N:C

N:N

Three Sites of Interaction During Assembly and Maturation

5

5 5

6

dsDNA Containing Phage Morphology

12

5

Scaffold Coat

Portal Minor Procapsid

Closure proteins Mature Virus

tailspike

The dsDNA Phage Assembly Pathway

Phi29 Scaffold Has a Helix-Loop-Helix Motif and a Disordered Tail

Marc C Morais et al., Nature Structural Biology 2003

Both Free and Bound Scaffold Exchange Bimodally

1 min

40 sec

0 sec

10 sec

10 min

3 min

857 858 859 860 861 862 863 864m/z0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

Free scaffold 13+

m/z

Partially exchanged species

Fully exchanged species

857 858 859 860 861 862 863 864m/z0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

Prohead scaffold

m/z

What does Bimodality Indicate? A group of residues open cooperatively & completely exchange before close again.

857 858 859 860 861 862 863 864m/z

1

4

2

3 slower components opened cooperatively

closed

exchanged

surface residues exchanged

1 2

4 4

3

The Bimodality Maps to N-terminal Helix-Loop-Helix

1.2min

40 sec

0 sec

20 sec

45 min

3.2min

R 20-31

m/z 687 688 689 690 691 692 693 694

691.92691.41

690.92690.40

692.42

692.92

691.91691.41

690.90690.41689.91

692.42

692.92 693.43

689.90689.41

688.906.40

688.42

690.41691.41 691.92

692.42

692.92 693.39

689.41

688.90

688.40

689.91

690.41691.42 691.91

692.42692.93 693.45

689.40688.90

688.39687.90

689.92

690.41 690.91691.41

687.39

687.89

687 688 689 690 691 692 693 694

m/z

1.2min

40 sec

20 sec

45 min

3.2min

Peptides Derived from H-L-H Region Have Similar Opening Kinetics

0 20 40 60 80 100 120 140 160 180 580 600

0

20

40

60

80

100

% c

ompl

etel

y ex

chan

ged

spec

ies

exchange time ( sec )

R 1-19 R 20-31 R 32-38 Whole protein

R 1-19

R 20-31

R 32-38

686 687 688 689 690 691 692 693

686 687 688 689 690 691 692 693

20 sec

3 min

5 min

30 hr

7 min

20 sec

3 min

1.5 hr

9 hr

30 hr

4.5 hr

m/z m/z

The Cooperative Motions can Be “Frozen” by Lowering the Temperature

20 °C 10 °C

Does Bimodality Originate from Opening of the Interface between Helices 1 & 2 ?

L3C & N35C

2.58 Å

Tethered Form

686 687 688 689 690 691 692 693 694

686 687 688 689 690 691 692 693 694

Reduced form

Oxidized Form ( Tethered )

The Tethered Form Cannot Open Cooperatively

5 min

2 min

10 sec

1 min

10 min

1 min

10 sec

20 sec

10 min

• Does Binding Alter the Exchange Kinetics?

686 687 688 689 690 691 692 693

686 687 688 689 690 691 692 693

0 50 100 150 200 250 2500 3000

0.0

0.2

0.4

0.6

0.8

1.0

% c

ompl

etel

y ex

chan

ged

sec

Free Prohead

Free Scaffold Prohead Scaffold

20 sec

40 sec

80 sec

45 min

200 sec

Prohead Bound Scaffold has Faster Kinetics

Model : The Interactions Stabilizing H-L-H are Weakened when Contact with Capsid