Protein structure at action: bind transform release презентация

BIND: repressors
α- turn - α

DNA & RNA
BINDING
Zn-
fingers
Leu-zipper

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

Immunoglobulin

Standard positions of active sites in protein folds

There are some
with catalytic
(Ser-protease) site

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

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

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

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

BIND ? TRANSFORM ? RELEASE: ENZYME
Note:
small
active
site
chymotrypsin

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

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

Chymotrypsin catalyses hydrolysis of a peptide
Spontaneous hydrolysis: very slow

SER-protease:
catalysis

CHYMOTRYPSIN ACTIVE SITE with INHIBITOR

Preferential binding of TS: RIGID enzyme
F = k1x1 = - k2x2 Ei = (ki /2)(xi)2 = F2/(2ki )
Hooke’s & 2-nd Newton’s Energy is concentrated
laws 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) = -(3πDη)v [Stokes law]
D – diameter; m ~ D3 – mass; η – viscosity
tkinet ≈ 10-13 sec × (D/nm)2 in water

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

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

MOTIONS

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

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

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

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

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)

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

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

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

Mechanochemical cycle
Myosin
Actin

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

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

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

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

SUMMARY
of the course

PROTEIN PHYSICS
Interactions
Structures
Selection
States & transitions

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

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…
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