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

BIND: repressors - turn - BIND: repressors - turn -

Znfingers DNA & RNA BINDING Leu-zipper Znfingers DNA & RNA BINDING Leu-zipper

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

Immunoglobulin Immunoglobulin

Standard positions of active sites in protein folds Standard positions of active sites in protein folds

There are some with catalytic (Ser-protease) site There are some with catalytic (Ser-protease) site

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

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

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

BIND TRANSFORM RELEASE Catalytic antibodies ABZYM = Anty. Body en. ZYM Transition state (TS) BIND TRANSFORM RELEASE Catalytic antibodies ABZYM = Anty. Body en. ZYM Transition state (TS) Antibodies are selected to TS-like molecule Preferential binding of TS: RIGID enzyme

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

Sometimes: Different folds with the same active site: the same biochemical function 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 nonactive POST-TRANSLATIONAL MODIFICATION Sometimes, only the CHAIN CUT-INDUCED DEFORMATION MAKES THE ENZYME ACTIVE READY nonactive “cat. Chymotripsinog site” en active cat. site Chymotripsin CUT

Chymotrypsin catalyses hydrolysis of a peptide Spontaneous hydrolysis: very slow Chymotrypsin catalyses hydrolysis of a peptide Spontaneous hydrolysis: very slow

SER-protease: catalysis SER-protease: catalysis

CHYMOTRYPSIN ACTIVE SITE with INHIBITOR CHYMOTRYPSIN ACTIVE SITE with INHIBITOR

Preferential binding of TS: RIGID enzyme F = k 1 x 1 = - Preferential binding of TS: RIGID enzyme F = k 1 x 1 = - k 2 x 2 Hooke’s & 2 -nd Newton’s laws Ei = (ki /2)(xi)2 = F 2/(2 ki ) 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 ~ D 3 – mass; – viscosity tkinet 10 -13 sec (D/nm)2 in water

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

MOTIONS MOTIONS

Double sieve: movement of substrate from one active site to another t. RNAIle Double sieve: movement of substrate from one active site to another t. RNAIle

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

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

Movement in quaternary structure: Hemoglobin vs. myoglobin noncovalent move of O 2 to and Movement in quaternary structure: Hemoglobin vs. myoglobin noncovalent move of O 2 to and from Fe needs fluctuation of a few protein’s side

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

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

Myosin Actin Mechanochemical cycle Myosin Actin Mechanochemical cycle

Rotary motor F 0 F 1 -ATP synthase structure from the X-ray data: Junge, Rotary motor F 0 F 1 -ATP synthase structure from the X-ray data: Junge, Sielaff, Engelbrecht, Nature, 459, 364 (2009)

Rotary motor F 0 F 1 -ATP synthase Basic side Acid side F 0 Rotary motor F 0 F 1 -ATP synthase Basic side Acid side F 0 -machine: H+-turbine Elston, Wang, Oster, Nature, 391, 510 Engelbrecht & Junge, FEBS Lett. 414, 485 (1997)

Rotary motor F 0 F 1 -ATP synthase working cycle of the H+-turbine Rotary motor F 0 F 1 -ATP synthase working cycle of the H+-turbine

H+ binding in Spirulina platensis H 3 O+ binding in Bacillus pseudofirmus Rotary motor H+ binding in Spirulina platensis H 3 O+ binding in Bacillus pseudofirmus Rotary motor Ion binding to the rotor ring of F 0 F 1 -ATP synthase Pogoryelov, Yildiz, Faraldo-Gómez, Meier, Nat. Struct. Mol. Biol. , 16, 1068 (2009) Preiss, Yildiz, Hicks, Krulwich, Meier, PLo. S Biol. 8, e 1000443 (2010)

SUMMARY SUMMARY

PROTEIN PHYSICS · Interactions · Structures · Selection · States & transitions PROTEIN PHYSICS · Interactions · Structures · Selection · States & transitions

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