PROTEIN PHYSICSPROTEIN PHYSICS

LECTURE 19LECTURE 19

Protein StructuresProtein Structures: Kinetic Aspects (1): Kinetic Aspects (1)

Basic facts on Basic facts on in vivo in vivo and and in vitro in vitro folding: folding: protein folds spontaneouslyprotein folds spontaneously

Levinthal paradox, or: Levinthal paradox, or: how can protein fold spontaneously?how can protein fold spontaneously?

Protein folding intermediatesProtein folding intermediates

Cunning simplicity hierarchic foldingCunning simplicity hierarchic folding

In vivoIn vivo (in the cell): (in the cell):

- RNA-encoded protein chain is synthesized at a - RNA-encoded protein chain is synthesized at a ribosome.ribosome.

- Biosynthesis + Folding < 10 – 20 min. - Biosynthesis + Folding < 10 – 20 min.

- Folding of large (multi-domain) protein: during the - Folding of large (multi-domain) protein: during the biosynthesis.biosynthesis.

- Folding is aided by chaperons and enzymes like - Folding is aided by chaperons and enzymes like disulfide isomerase. disulfide isomerase.

- The main obstacle for The main obstacle for in vivo in vivo folding experiments:folding experiments:

nascent protein is small, ribosome (+ …) is large. nascent protein is small, ribosome (+ …) is large.

BASIC FACTS:BASIC FACTS:

The main obstacle for The main obstacle for in vivo in vivo folding experiments:folding experiments:

nascent protein is small, ribosome (+ …) is large. nascent protein is small, ribosome (+ …) is large.

However, one can follow some “rare” protein activity,However, one can follow some “rare” protein activity,

and use a “minimal” cell-free systemand use a “minimal” cell-free system

Luciferase activityLuciferase activity

(Kolb, Makeev,(Kolb, Makeev,Spirin, 1994)Spirin, 1994)

In vitroIn vitro (in physico-chemical experiment):(in physico-chemical experiment):

-Unfolded globular Unfolded globular protein is capable of protein is capable of renaturationrenaturation(if it is not too large and not too modified chemically after (if it is not too large and not too modified chemically after the biosynthesis), i.e., its 3D structure is capable ofthe biosynthesis), i.e., its 3D structure is capable of spontaneous folding [Anfinsen, 1961].spontaneous folding [Anfinsen, 1961].

- Chemically synthesized protein chain achieves its - Chemically synthesized protein chain achieves its correct 3D structure [Merrifield, 1969]. correct 3D structure [Merrifield, 1969].

- The main obstacle for - The main obstacle for in vitro in vitro folding is aggregation. folding is aggregation.

BASIC FACTS:BASIC FACTS:

ConclusionConclusion: Protein structure is determined by its amino : Protein structure is determined by its amino acid sequence; acid sequence; cell machinery is not more than an “incubator” for protein cell machinery is not more than an “incubator” for protein folding. folding.

HOW DOES PROTEIN FOLD?HOW DOES PROTEIN FOLD?and even moreand even more::How CAN protein fold spontaneously?How CAN protein fold spontaneously?

Levinthal paradox (1968):Levinthal paradox (1968):

FOLDING INTERMEDIATES??FOLDING INTERMEDIATES??

Native protein structure Native protein structure reversibly refolds from reversibly refolds from various starts, i.e., it is various starts, i.e., it is thermodynamically thermodynamically stable.stable.

But how can protein But how can protein chain find this unique chain find this unique structure - within structure - within seconds - among zillions seconds - among zillions alternatives?alternatives?

““Framework model” of Framework model” of stepwisestepwise folding folding(Ptitsyn, 1973)(Ptitsyn, 1973)

Now: Now: Pre-molten Pre-molten globule globule

Now: Now: Molten Molten globuleglobule

Kinetic intermediate (molten globule) in protein foldingKinetic intermediate (molten globule) in protein folding

(Doldikh,…, Ptitsyn, 1984)(Doldikh,…, Ptitsyn, 1984)

Multi-state foldingMulti-state folding

LAG

Extensive search for metastable (accumulating, directly observable) folding intermediates: MG-like, with partly formed S-S bonds, etc.The idea was that the intermediates, if trapped, would help to trace the folding pathway, like intermediates in a biochemical reaction trace its pathway. This was, as it is now called, “chemical logic”.

However, although protein folding intermediates (like MG) were found for many proteins, the main question as to how the protein chain can find its native structure among zillions of alternatives remained unanswered. 

Cunning simplicity of hierarchic foldingCunning simplicity of hierarchic folding as applied to resolve the Levinthal paradoxas applied to resolve the Levinthal paradox

Folding intermediates Folding intermediates must become more and more stable for hierarchic folding. must become more and more stable for hierarchic folding. This cannot provide This cannot provide a simultaneousa simultaneous explanation to explanation to (i)(i) folding within non-astronomical time;folding within non-astronomical time;(ii)(ii) ““all-or-none” transition, i.e., co-existence of only native all-or-none” transition, i.e., co-existence of only native

and denatured molecules in visible amount; and denatured molecules in visible amount; (iii)(iii) the same 3D structure resulting from different pathwaysthe same 3D structure resulting from different pathways

hierarchic hierarchic (stepwise)(stepwise)foldingfolding

All-or-none All-or-none transition:transition:

In thermo-In thermo-dynamicsdynamics

In kineticsIn kinetics

…folding intermediates were found, but the main question as to how the protein chain can find its native structure among zillions of alternatives remained unanswered. 

A progress in the understanding was achieved when the studies involved small proteins (of 50 - 100 residues). Many of them are “two-state folders”: they fold in vitro without any observable (accumulating) intermediates, and have only two observable states: the native fold and the denatured coil.