PROTEIN PHYSICSPROTEIN PHYSICS

LECTURE 24-25LECTURE 24-25

PROTEIN STRUCTURE AT ACTION:PROTEIN STRUCTURE AT ACTION:

BIND BIND TRANSFORM TRANSFORM RELEASE RELEASE

BIND: repressorsBIND: repressors

-- turnturn --

DNA & RNADNA & RNABINDINGBINDING

Zn-Zn-fingersfingers

Leu-zipperLeu-zipper

-BINDING-INDUCED DEFORMATION -BINDING-INDUCED DEFORMATION MAKES REPRESSOR ACTIVE, and IT BINDS TO DNAMAKES REPRESSOR ACTIVE, and IT BINDS TO DNA

BIND BIND RELEASE: RELEASE: REPRESSOR

ImmunoglobulinImmunoglobulin

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

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

Preferential binding of TS: Preferential binding of TS: RIGID RIGID enzymeenzyme

CatalysisCatalysis:: stabilization of the transition state stabilization of the transition state (TS) (TS)

Theory: Pauling & HoldenTheory: Pauling & Holden

BINDBIND TRANSFORM TRANSFORM RELEASERELEASE

Catalysis: stabilization of the transition stateCatalysis: stabilization of the transition state (TS) (TS)

Theory: Pauling & HoldenTheory: Pauling & HoldenExperimental verification: FershtExperimental verification: Fersht

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reputedreputedTSTS

Catalysis: stabilization of the transition stateCatalysis: stabilization of the transition state (TS) (TS)

Theory: Pauling & HoldenTheory: Pauling & HoldenExperimental verification: FershtExperimental verification: Fersht

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reputedreputedTSTS

This This proteinproteinengineeringengineeringreducesreducesthe ratethe rateby 1000000by 1000000

PreferentialPreferentialbinding binding of TS:of TS:RIGIDRIGID

enzymeenzyme

Catalytic antibodiesCatalytic antibodies ABZYM = AABZYM = AntyntyBBodyody enenZYMZYM

AntibodiesAntibodiesare are

selectedselectedto TS-liketo TS-likemoleculemolecule

Transition state (TS)Transition state (TS)

PreferentialPreferentialbinding binding of TS:of TS:RIGIDRIGID

enzymeenzyme

BIND BIND TRANSFORMTRANSFORM RELEASE RELEASE

BIND BIND TRANSFORM TRANSFORM RELEASE RELEASE:: ENZYME

Note: small active site

chymotrypsinchymotrypsin

SometimesSometimes::

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

POST-TRANSLATIONAL MODIFICATIONPOST-TRANSLATIONAL MODIFICATION

Sometimes, Sometimes, only theonly the CHAIN CUT-INDUCED DEFORMATION CHAIN CUT-INDUCED DEFORMATION MAKES THE ENZYME ACTIVE READYMAKES THE ENZYME ACTIVE READY

ChymotripsinoChymotripsinogengen

active cat. site

non-active “cat. site” CUT

ChymotripsinChymotripsin

Chymotrypsin catalyses hydrolysis of a peptide Chymotrypsin catalyses hydrolysis of a peptide

Spontaneous hydrolysis: very slowSpontaneous hydrolysis: very slow

SER-protease: SER-protease: catalysiscatalysis

CHYMOTRYPSIN ACTIVE SITECHYMOTRYPSIN ACTIVE SITE with with INHIBITOR INHIBITOR

Preferential binding of TS: Preferential binding of TS: RIGID RIGID enzymeenzyme

F F = = kk11xx11 = - = - kk22xx22 E Ei i = (= (kkii /2/2)()(xxii))22 = = FF22//((2k2kii ))Hooke’s & 2-nd Newton’s Energy is concentrated Hooke’s & 2-nd Newton’s Energy is concentrated laws in the laws in the softersofter body. body. Effective catalysis: when Effective catalysis: when substrate is softer than substrate is softer than proteinprotein

Kinetic energy Kinetic energy cannot be stored cannot be stored for catalysisfor catalysisFriction stops a molecule within Friction stops a molecule within picoseconds: picoseconds:

m(dv/dt) = -(3m(dv/dt) = -(3DD)v )v [Stokes law][Stokes law]D – diameter; m ~ DD – diameter; m ~ D33 – mass; – mass; – viscosity – viscosity

ttkinetkinet 10 10-13 -13 sec sec (D/nm) (D/nm)22 in waterin water

PROTEIN STRUCTURE AT ACTION:PROTEIN STRUCTURE AT ACTION:

BIND BIND TRANSFORM TRANSFORM RELEASE RELEASE

RIGID CATALITIC SITERIGID CATALITIC SITE

INDEPENDENT ON OVERALL CHAIN FOLDINDEPENDENT ON OVERALL CHAIN FOLD

MOTIONSMOTIONS

Double sieve:Double sieve: movement of substrate movement of substrate

from one active site to anotherfrom one active site to another

tRNAtRNAIleIle

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

another for catalysisanother for catalysis

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

Individual substrate-binding domainIndividual substrate-binding domain

Movement in quaternary structure: Movement in quaternary structure: Hemoglobin vs. myoglobinHemoglobin vs. myoglobin

Механохимический цикл

МиозинМиозин АктинАктин

АТФ АДФ + Ф15 ккал/мольв клеточных

условиях

Mechanochemical cycleMechanochemical cycle

MyosinMyosin

ActinActin

SUMMARYSUMMARY

PROTEIN PHYSICSPROTEIN PHYSICS

InteractionsInteractions

StructuresStructures

SelectionSelection

States & States & transitionstransitions

Intermediates Intermediates & nuclei& nuclei

Structure Structure prediction & prediction & bioinformaticsbioinformatics

Protein Protein engineering & engineering & designdesign

Functioning Functioning