6. Folding and flexibility

By Assist. Prof. Betul Akcesme

• “Prediction of three dimensional structure of a protein from its amino acid sequence is the major unsolved problem in structural molecular biology”

Introduction

a polypeptide chain acquires its correct 3D structure to achieve the biologically

active native state

PROTEIN FOLDING

Some proteins folds spontaneously, others require assistance such as enzymes ,chaperones)

CHAPERONS 1.Binds to partially folded polypeptide prevents making unwanted bonding - promotes the folding 2. After a sequence acquires most of its correct secondary structure, it has a looser tertiary structure 3. FINAL native state

MOLTEN GLOBULAR STATE

Molten globular state to native state spontaneously

How to predict the 3D structure from primary structure?

native state protein is not static:

secondary elements, domains undergo small movements in space:

individual atoms or collectively;

Functional activities of protein : ligand binding trigger

conformation changes.

Major unsolved problem

Globular proteins are only

marginally stable

Two major contributors to the energy difference: Enthalpy and Entropy

Native state (folded) Denatured state (unfolded)

Energy difference: 5 – 15 kcal/mol (a single H-bond: 2 – 5 kcal/mol)

Decrease for spontaneous process

energy of noncovalent interactions including the hydrophobic interactions, hydrogen bonds, and ionic bonds. These interactions are maximized to produce globular molecule in native state Covalent interactions ? These non‐covalent interactions are therefore stronger and more frequent in the native state and hence their energy contribution, enthalpy, is much larger. The enthalpy difference between native and denatured states can reach several hundred kcal/mol.

native state is order, denatured states is disorder –each denatured structure is unique.

ENTHALPY

ENTROPY

Denatured state Native state

entropy between the native ordered state and the denatured state can also reach several hundred kcal/mol but in the opposite direction to the enthalpy difference.

FREE ENERGY difference between states are very small!

• Fig.6.22 from Mathews, van Holde & Ahern, Biochemistry, 3rd ed. (2000): Thermodynamics

of protein folding

• summary of overall interplay between DH and the -TDS components (both hydrophobic effect and

conformational entropy)

• A simple reaction profile for two state reaction. The reaction can be

described in terms entirely analogous to reaction kinetics where G is

the conformational stability of the folded protein (GU − GF). For

folding to occur the change in free energy must be negative

The marginal stability of the native state is biologically

and functionally important!

Globular proteins in correct quantities at appropriate times.

THEREFORE

It is equally important to be able to degrade proteins as it is to be able to synthesize them. Globular proteins in living cells usually have a rapid turnover ,which means their native states is marginally stable.

Some enzymes need structural flexibility, which would be inconsistent with a rigidly stabilized structure.

Kinetic factors are important for folding

“Calculation for searching all possible conformation in a

random fashion to get the lowest energy is impossible!”

Assuming each peptide has only three (α,β,L) conformation and time scale is one pico-second (10-12)

(for a polypeptide chain of 150 amino acid residues)

3150 (=1068) possible conformations Time required is1068 x 10-12 = 1056 seconds (= 1048 years) cf) actual folding time: 0.1–1000 seconds both in vivo & in vitro

To occur on this short time scale, the folding process must be directed in some way through a “kinetic pathway of unstable intermediates ” to escape sampling a large number of irrelevant conformations

Levinthal Paradox

Kinetic trapping in a wrong minimum

• Protein might be trapped in a local low energy state with a high energy

barrier that prevents it from reaching the GLOBAL ENERGY

MINIMUM. ( give different fold)

• Structure prediction with energy calculation is (might) WRONG!

Kinetic factors are important for folding process it is possible that the

observed folded conformation is not the one with lowest free energy but

rather the most stable of those conformations that are kinetically accessible!

HOW A LIVING CELL CAN PREVENT THE FOLDING PATHWAY

FROM BECOMING BLOCK AT THE INTERMEDIATE STAGE?

The most common obstacles to correct folding

(1) Aggregation of the intermediates through exposed hydrophobic groups

(2) Formation of incorrect disulfide bonds

(3) Isomerization of proline residues

Can be overcome by CHAPERONES!

Needs “Special proteins that assist the folding protein – chaperone”

• Energy minima in protein

folding.

• Anfinsen’s thermodynamic

hypothesis suggests that the three-

dimensional structure of a native

protein under physiological

conditions is one in which the

free energy of the system is

the lowest (Anfinsen, C.

B. Science 1973,181, 223–230).

Thus, while proteins may sample

other local minima during

folding, there is only one true

minimum-energy fold.

Alternative way to remove kinetic barrier

• α lytic protease (bacterial enzyme)

• It is synthesized and folded in vivo but not active protein with

prosegment of 77 residues

This segment is excised after folding to PRODUCE ACTIVE ENZYME.

ADDING PROSEGMENT can induce refolding

Unfolded α lytic protease without this segment can not refold easily

The capacity of folding exits in unfolded enzyme BUT there is a BARRIER present

in folding pathway.

PROSEGMENT REMOVES THIS KINETIC BARRIER by lowering the free energy

of transition state for folding ( like enzyme catalyzes chemical reactions)

Within a few milliseconds also some native like alpha helix and beta strand positions

Up to 1 second

Molten globules are intermediates in folding

driving force not very known BUT • packing of core not formed •Interior side chains are mobile •Loops and surface elements remain unfolded

Ensemble of conformations are reduced

single native form is reached, forming native interactions, including hydrophobic packing in the interior & fixing surface loops

UNFOLDED MOLTEN GLOBULE

FOLDED

Folding occurs through

the progressive

organization of

ensembles of structures

[shown here for the src-

SH3 domain (left)] on a

funnel-shaped Free

energy landscape

(right).

Conformational

entropy loss during

folding is compensated

by the free energy

gained as more Native

interactions are formed.

Kinetics is determined by the local roughness of the landscape, relative to thermal energy. Key interactions in early folding (dashed circle) coincide, for this protein, with experimentally determined regions.

Main mystery of protein folding is…

…The collapse of the unfolded state to generate

the molten globule embodies

• WHAT IS THE DRIVING FORCE BEHIND THE CHOICE OF NATIVE

TERTIARY FOLD FROM A RANDOMLY ORIENTED POLPEPTIDE CAHIN?

1. Secondary structure formation cannot be the thermodynamic driving force of protein folding…

BECAUSE stable hydrogen bonds can also be formed to water molecules in the unfolded state. (There is very little change in Free Energy)

2. Large Free energy change by bringing hydrophobic side chains out of contact with water and into contact with each other in the interior of a globular entity.

Burying hydrophobic side chains is a key event

Most likely scenario that the polypeptide chain begins to form compact shape

with Hydrophobic side chains partially buried very early in the folding…..

• This scenario has several important consequences

▫ Thus it vastly reduces the number of possible conformations that need

to be searched because only those that are sterically accessible within

this shape can be sampled.

▫ Furthermore, when side chains are buried, their polar backbone –NH and

–CO groups are also buried in a hydrophobic environment, hence unable to

form hydrogen bonds to water –

BUT they bond to each other – so you get alpha and beta

structures.

CONCLUSION Secondary structure elements NOT DRIVING FORCE for the formation of Molten Globule But they are consequences of burying hydrophobic side chains…

Core Questions

• Looking at the protein sequences of globular proteins, one finds that

hydrophobic side chains are usually scattered along the entire sequence,

seemingly randomly.

• In the native state of folded protein, ½ of these side chains are buried, and

the rest are scattered on the surface of the protein, surrounded by

hydrophilic side chains.

• The buried hydrophobic side chains are not clustered in the sequence.

• What causes these residues to be selectively buried during the

early and rapid formation of the molten globule?

• If we wish to solve the folding problem, this must be answered first.

The unfolded state consists of many

rapidly interconverting

conformationally different molecules,

U1...Un.

The molten globule is an ensemble of

structurally related molecules, M1...Mm,

which are rapidly interconverting and

which slowly change to a single unique

conformation, the folded state F.

The conversion from the molten globule

state to the folded state is slow and passes

through a high energy transition state, T.

High energy unfolded state

Low energy folded state

Both single and multiple folding pathways have been

observed

Both single and multiple folding pathways have been

observed

• Ui‘s --- unfolded states, many of them.

• Mi’s --- molten globule states. Has most secondary structures, but

less compact.

• Converging to F. During this relatively slower process it passes a

high energy transition state T.

• These facts have been verified by NMR, hydrogen exchange,

spectroscopy, and thermo-chemistry.

•

• Alan Fersht, Cambridge

• The techniques is based on investigation of the effects on the

energetics of folding of single mutation in a protein known

structure

EXAMPLE

Mutation ALA to GLY in solvent exposed face at an

alpha helix ,

both destabilize both intermediate state and

the native state, as well as transition state

the helix is already formed in the intermediate state

On the other hand, mutations destabilize the

native state but do not affect the energy of the

intermediate or transition state at all

the helix is formed after the transition state.

The story of barnase 110 amino acids, no disulfide bridges.

3 amino terminal alpha helices, and C terminal 5-

stranded antiparallel sheet.

Effects of all mutations are examined,

detailed residue characterization : its

folding intermediates and transition states

Folding process:

intermediate molten globule state: already

with most of native secondary

structures, and native like relative

position of alpha helix and beta sheet, as

well as relative positions of beta strands

within the sheet.

Single Major transition state! One major pathway !

The story of lysozyme

Hen egg-white lysozyme

The native structure has 2 lobes, the first

one with 5 alpha helices, the second is

mainly a 3-stranded antiparallel beta

sheet.

Folding is studied by NMR, Circular dichronism,

fluorescence, hydrogen- deuterium exchanges

Folding process: At 20 milliseconds, two

intermediate states of lysozyme were

detected: one with alpha domain formed, no

beta, the other with neither; in fact a third

(less popular) with both alpha and beta

domains developed.

But is this just because some folds slower? 2 major and one minor pathways!

The most common obstacles to correct folding

(1) Aggregation of the intermediates through exposed hydrophobic

groups

(2) Formation of incorrect disulfide bonds

(3) Isomerization of proline residues

Can be overcome by CHAPERONES!

Needs “Special proteins that assist the folding protein – chaperone”

• ENZYMES THAT HAVE ROLE IN FOLDING!

Enzymes assists formation of proper disulfide bonds during

folding

• Unfold proteins have no disulfide bonds.

• Formation of disulfide bonds require enzyme to help (oxidation of cysteine

residues).

▫ In bacteria, periplasmic space

Catalyzed by disulfide bridge forming enzyme (Dsb)

▫ In eukaryotes, disulfide bond formation occurs in E.R. before

transport to cell surface

▫ (not found in cytosol because of reducing environment but IN membrane or

secreted)

Protein disulfide isomerase(PDI)

Catalyze internal disulfide exchange to remove folding

intermediates with incorrectly formed disulfide bridge.

• Thomas Creighton,EMBL

• Peter Kim, MIT

• 58 residues.

• 6 cysteine residues which form 3 disulfide bonds.

• 30-51

• 5-14

• 14-38

• The influence of disulfide bond formation on folding!

• Folding pathways!

Bovine pancreatic trypsin inhibitor (BPTI)

Bovine pancreatic trypsin inhibitor (BPTI)

• Trapping disulfide bonded intermediates as a method for studying the folding pathways.

First bond randomly form … with 30-51 being more stable, 60%, Second bond (if the first is 30-51): 5-14, 5-38, 14-38 all possible. 14-38 native, others unfold. Formation of 5-55 occurs very slowly- buried inside the folded intermediate and not accessible to oxidizing agents

• Adding the enzyme protein disulfide isomerase significantly increases the rate of folding of protein BPTI.

• Prokaryote: Dsb (similar to thioredoxin)

• Eukaryote: PDI (Protein disulfide isomerase)

• Reduced (SH-containing) PDI catalyzes the rearrangement of a

polypeptide’s non-native disulfide bonds via disulfide interchange

reactions to yield native disulfide bonds.

• (b) Oxidized (disulfide-containing) PDI catalyzes the initial formation of a

polypeptide’s disulfide bonds through the formation of a mixed disulfide.

Reduced PDI can then react with a cellular oxidizing agent to regenerate

oxidized PDI.

Isomerization of proline residues

• Trans‐peptide with C=O and –NH groups pointing in opposite directions was

1000 times preferred to the cis‐peptide.(C=O and –NH groups pointing in

same directions)

• However, when the 2ndresidue is a proline the cis conformer is only 4 times

less stable. cis=-proline peotide in many proteins

• Mainly the Pro trans conformer,(few steric collusions) but the cis version is

found in tight turns and are sometimes essential for conformational flexibility.

• In the native protein the cis‐proline arrangements are stabilized by

tertiary structure interactions, but in the unfolded state there is an

equilibrium between cis/trans isomers.

• When refolding occurs, you can get the proline‐peptide bond in the wrong

form. From a kinetic standpoint, cis‐trans proline isomerization is a very

slow process that can prevent the progress of protein folding by trapping one

or more proline residues crucial for folding in the non‐native isomer,

especially when the native protein requires the cis isomer.

• More prolines—more chance. As noted…cis‐trans isomerization is a slow

process and in vitro is often the rate limiting folding step.

•NEED to get things in right conformations!

• Cis –trans isomerization of

proline peptides is a slow

process and in vitro it is

frequently the rate limiting

step in folding for those

molecules that have been

trapped in a folding

intermediate with wrong

isomers.

• In vivo, rates of this process are enhanced by enzymes initially

called peptidyl prolyl isomerases.

• First one found cyclophilin

• *impacts the rate of cis‐trans isomerization of proline peptides by a factor of

a million over non enzmatic reaction Invloved in immunosupression by inhibiting T cell proliferation after binding immunosupressive drugs ( unrelated to their isomerase activity) Graft rejection! (involved in immunosupression)

Proline containing tetrapeptide

The active site is on the outside of beta barrel

Anfinsen's dogma (also known

as the thermodynamic

hypothesis) is a postulate in

molecular biology championed

by the Nobel Prize winner

Christian B. Anfinsen.

1) CAN A PROTEIN FOLD SPONTANEOUSLY TO ITS NATIVE STATE?

YES !

How the protein reaches this structure is

really the subject of the field of protein

folding, which has a related dogma called

Levinthal's paradox.

The Levinthal paradox states that the number of possible

conformations available to a given protein is astronomically

large, such that even a small protein of 100 residues would

require more time than the universe has existed to explore

all possible conformations and choose the appropriate one,

it would also arguably make computational prediction of

protein structures under the same basis unfeasible if not

impossible.

2) DOES A PROTEIN SEARCH ALL POSSIBLE OUTCOMES TO FIND THE FINAL CONFORMATION , THE ONE OF LOWEST FREE ENERGY?

NO!

Anfinsen Experiment

Protein Folding

• Protein folding considers the question of how

the process of protein folding occurs, i. e.

• unfolded native state.

• Importance:

• – Predict 3D structure from primary sequence

• – Avoid misfolding related to human diseases

• – Design proteins with novel functions

ANFINSEN: AMINO ACID SEQUENCE DETERMINES PROTEIN SHAPE

The dogma states that, at least for small globular proteins, the native

structure is determined only by the protein's amino acid sequence.

At the environmental conditions (temperature, solvent concentration and

composition, etc.) at which folding occurs,

the native structure is a unique, stable and kinetically accessible minimum of

the free energy.

Hypothesis

“protein amino acid sequence determines the final

shape a protein assumes in a water solution”

UNFOLDING RIBONUCLEASE

• Ribonuclease

▫ the hydrolysis of RNA, and its enzymatic activity depends

entirely upon the protein being in a particular shape.

• 124‐amino acid residues. 4 disulfide bridges

• Bonds can be reduced (electrons removed) with high

concentrations of the sulfhydryl reagent b-

mercaptoethanol

–S—S—becomes –SHHS—

• Altering the polar nature of the solvent by adding

urea,

▫ the reduced ribonuclease, lacking the disulfide bonds to

resist the stress, open up (denatures) into a random

coil that has no enzyme activity.

1st experiment

• Free of the reducing agent, the sulfhydryl groups (--SH) of the

cysteines were being oxidized by dissolved oxygen from the air, and

the protein was refolding into the catalytically active shape.

• This could only mean that the folding was indeed directed by the

amino acid sequence

When Anfinsen did this, he observed that the ribonuclease protein slowly regained its enzymatic activity.

1st experiment

Anfinsen Experiment

• Remove β-mercaptoethanol only,

oxidation of the sulfhydryl group,

then remove urea → scrambled

protein, no activity

• Further addition of trace amounts

of β-mercaptoethanol converts the

scrambled form into native form.

• Conclusion: The native form of a

protein has the

thermodynamically most stable

structure.

2nd experiment

3rd experiment

2nd experiment

1st experiment

3rd experiment

Conclusion

• The original work led Anfinsen to propose his "Thermodynamic

Hypothesis", which states that the native conformation of a

protein is adopted spontaneously.

• In other words, there is sufficient information contained in

the protein sequence to guarantee correct folding from any

of a large number of unfolded states.

• This demonstrated that the shape that a protein realizes in solution

is dictated by amino acid sequence information, which is expressed

in terms of thermodynamic stability

Protein Folding Pathway