Protein structure at action: bind transform release

1.

PROTEIN PHYSICS
LECTURE 24-25
PROTEIN STRUCTURE AT ACTION:
BIND TRANSFORM RELEASE

2.

BIND: repressors
- turn -

3.

Znfingers
DNA & RNA
BINDING
Leu-zipper

4.

BIND RELEASE: REPRESSOR
-BINDING-INDUCED DEFORMATION
MAKES REPRESSOR ACTIVE, and IT BINDS TO DNA

5.

Immunoglobulin

6.

Standard positions of active sites
in protein folds

7.

There are some
with catalytic
(Ser-protease) site

8.

BIND TRANSFORM RELEASE
Catalysis:
stabilization of the transition state (TS)
Theory: Pauling & Holden
Preferential binding of TS: RIGID enzyme

9.

Catalysis: stabilization of the transition state (TS)
Theory: Pauling & Holden
Experimental verification: Fersht
reputed
TS
__________
P
______

10.

Catalysis: stabilization of the transition state (TS)
Theory: Pauling & Holden
Experimental verification: Fersht
reputed
TS
/
/
__________
P
/
/
______
This
protein
engineering
reduces
the rate
by 1000000
Preferential
binding
of TS:
RIGID
enzyme

11.

BIND TRANSFORM RELEASE
Catalytic antibodies
ABZYM = AntyBody enZYM
Suggested by Jencks in 1969
Done by Schultz and Lerner in 1994
Transition state (TS ‡)
Antibodies
are
selected
to TS-like
molecule
Preferential
binding
of TS:
RIGID
enzyme

12.

BIND TRANSFORM RELEASE: ENZYME
chymotrypsin
Note:
small
active
site

13.

Sometimes:
Different folds with the same active site:
the same biochemical function

14.

POST-TRANSLATIONAL MODIFICATION
Sometimes, only the CHAIN CUT-INDUCED DEFORMATION
MAKES THE ENZYME ACTIVE READY
non-active
“cat. site”
Chymotripsinogen
active
cat. site
Chymotripsin
CUT

15.

Chymotrypsin catalyses hydrolysis of a peptide
Spontaneous hydrolysis: very slow

16.

SER-protease:
catalysis

17.

CHYMOTRYPSIN ACTIVE SITE with INHIBITOR

18.

Preferential binding of TS: RIGID enzyme
F = k1x1 = - k2x2
Hooke’s & 2-nd Newton’s
laws
Ei = (ki /2)(xi)2 = F2/(2ki )
Energy is concentrated
in the softer body.
Effective catalysis: when
substrate is softer than protein
Kinetic energy cannot be stored for catalysis
Friction stops a molecule within
picoseconds:
m(dv/dt) = -(3 D )v [Stokes law]
D – diameter; m ~ D3 – mass; – viscosity
tkinet 10-13 sec (D/nm)2
in water

19.

PROTEIN STRUCTURE AT ACTION:
BIND TRANSFORM RELEASE
RIGID CATALITIC SITE
INDEPENDENT ON OVERALL CHAIN FOLD

20.

Lock and key
model
for enzyme catalysis.
Hermann Emil Louis
Fischer (1852 –1919)
Induced fit
model
for enzyme catalysis.
Daniel Edward Koshland, Jr.
(1920 – 2007)

21. MOTIONS

22.

Double sieve:
movement of substrate
from one active site to another
tRNAIle
Fersht A.R., Dingwall C. (1979)

23.

Movement in two-domain enzyme:
One conformation for binding (and release),
another for catalysis
Induced fit

24.

Two-domain dehydrogenases:
Universal NAD-binding domain;
Individual substrate-binding domain

25.

Movement in quaternary structure:
Hemoglobin vs. myoglobin
non-covalent
bonding of O2
move of O2 to and from Fe needs
fluctuation of a few protein’s side chains

26.

Kinesin : Linear cyclic motor
the simplest one-direction walking machine with cyclic ligand-induced
conformational changes and bindings/unbindings to tubulin microtubule
Mandelkow & Mandelkow,
Trends Cell Biol. 12, 585 (2002)
The head “feels” its position, front or rear,
due to its interaction with the linker. Yildiz, Tomishige, Gennerich, Vale, Cell 134, 1030 (2008)

27.

Kinesin : Linear cyclic motor
the simplest one-direction walking machine with cyclic ligand-induced
conformational changes and bindings/unbindings to tubulin microtubule

28.

Sir Andrew Fielding Huxley
(1917 – 2012)
Nobel Prize 1963
Myosin "cross-bridges"

29.

Миозин
Актин
АТФ АДФ + Ф
15 ккал/моль
в клеточных
условиях
Механохимический цикл

30.

Myosin
Actin
Mechanochemical cycle

31.

Rotary motor
F0F1-ATP synthase
structure from the X-ray data: Junge, Sielaff, Engelbrecht, Nature, 459, 364 (2009)

32.

Rotary motor
F0F1-ATP synthase
Basic side
Acid side
F0-machine: H+-turbine
Elston, Wang, Oster, Nature, 391, 510 (1998)
Engelbrecht & Junge, FEBS Lett. 414, 485 (1997)
Elston, Wang, Oster, Nature, 391, 510 (1998)

33.

Rotary motor
F0F1-ATP synthase working cycle of the H+-turbine

34.

H+ binding in Spirulina platensis
H3O+ binding in Bacillus pseudofirmus
Rotary motor
Ion binding
to the rotor ring of
F0F1-ATP synthase
Pogoryelov, Yildiz,
Faraldo-Gómez, Meier,
Nat. Struct. Mol. Biol.,
16, 1068 (2009)
Preiss, Yildiz, Hicks,
Krulwich, Meier,
PLoS Biol. 8,
e1000443 (2010)

35.

SUMMARY
of the course

36.

PROTEIN PHYSICS
Interactions
Structures
Selection
States &
transitions

37.

Intermediates
& nuclei
Structure
prediction &
bioinformatics
Protein
engineering &
design
Functioning

38.

Благодарю за внимание
…
товарищи офизевшие биологи!

39.

Благодарю за внимание
…
товарищи офизевшие биологи!