September 15, 2003 Lecture 6/MBB 222 1

Any peptide (in theory) could adopt many different secondary and tertiary structures

But in general

ALL molecules of a given protein species adopt the SAME 3D-conformation. This structure is called the NATIVE STATE of the protein

- the native state is usually (but not always) the most stable (lowest energy) state of the folded protein

- disruption of the native structure (by BREAKING the weak bonds responsible for 2° and 3° structures) is called denaturation

- the result is a protein in a non- native or denatured state

Denaturation can be caused by:- raising (or more rarely lowering) the temperature- extremes of pH-chaotropes (such as 8M urea or guanidine hydrochloride)- detergents etc.

Most denatured proteins precipitate

Protein folding:Protein folding:the native and non-native statesthe native and non-native states

September 15, 2003 Lecture 6/MBB 222 2

Denatured proteins will spontaneously refold Denatured proteins will spontaneously refold inin vitrovitro ((in the test tube) e.g. folding of RNAse A in the test tube) e.g. folding of RNAse A

denaturation renaturation

Incubate proteinin guanidine

hydrochloride(GuHCl)or urea

100-folddilution of proteininto physiological

buffer

Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230.

- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation

Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors”

September 15, 2003 Lecture 6/MBB 222 3

Protein Folding: the Levinthal paradoxProtein Folding: the Levinthal paradox

folding

denaturedprotein:random coil-a very large number of possible extendedconformations

native protein1 stable

conformation

in vitro in vivo

folding

t = seconds t = seconds or less

September 15, 2003 Lecture 6/MBB 222 4

Levinthal paradox: a new folding view is needed

Levinthal paradox: a new folding view is needed

Consider a protein with 100 amino acids

- using a very simplified model where there are only 3 possible orientations per residue

- assume 1 conformation can form every 10-13 seconds

(100 picoseconds)

- then 5 x 1047 x 10-13 s = 1.6 x 1027 years to correctly fold a protein

Obviously NOT ALL conformations can be ‘sampled’ during folding!

3100 different conformations = 5 x 1047 !!!

September 15, 2003 Lecture 6/MBB 222 5

Resolving the paradoxResolving the paradoxa. there are a limited number of secondary structural elements

b. these elements tend to form spontaneously during the co-translational folding of a protein

c. proteins fold via so-called “folding landscapes”, where the proteins follow “pathways” of folding that lead to the correct three-dimensional structure

d. folding intermediates may be important in such folding landscapes/pathways

September 15, 2003 Lecture 6/MBB 222 6

• folding can be thoughtfolding can be thoughtto occur alongto occur along““energy surfaces or energy surfaces or landscapes”landscapes”

• limited number of limited number of secondary structure secondary structure elements: helices,elements: helices,sheets and turnssheets and turns

Protein folding theoryProtein folding theory

Dobson, CM (2001)Phil Trans R Soc Lond 356, 133-145

September 15, 2003 Lecture 6/MBB 222 7

A simplified view: A folding funnelA simplified view: A folding funnel

unfolded (non-native) states(two of many different conformations are shown)

Native state (N)

Energy landscape- descent towards Free energy minimum state

- A- rapid folding B- secondary energy minima

September 15, 2003 Lecture 6/MBB 222 8

Usually, ∆G for folding is negative i.e. favourable; but this is due to a balance of several thermodynamic factors:-Conformational entropy: works against folding (contributes positively to ∆ G), since unfolded condition = random cycling between many possible states, it involves higher entropy than the single folded state.

-Enthalpy contribution: works in favour of folding (contributes negatively to G) i.e. reduction in Enthalpy due to formation of energetically favourable interactions (e.g. salt bridges, H bonds, van der Waals etc.) between chemical groups in folded state.

-Entropy contribution from hydrophobic effect: works in favour of folding (contributes negatively to G) i.e. the burying of hydrophobic R groups in protein increases entropy of whole system (protein + water).

∆G = ∆H - T∆S

overall free energy change for folding

Thermodynamics of protein folding

balance = -ve ∆ G

September 15, 2003 Lecture 6/MBB 222 9

Proteins fold in stagesProteins fold in stages

Local folding through nucleation of small clusters of residues

A General Order of Folding1. Short regions rapidly form small stable secondary elements; like alpha-

helices and beta-sheets etc.2. These small structure elements interact with other local elements and

interact folding into ‘globular’ units on an intermediate time scale. Associations are through various weak chemical interactions.

3. These globular domains may be a complete small protein or a number of these in larger proteins can interact more slowly to fold into the final folded structure.

polypeptide foldedprotein

secondary structures domains

September 15, 2003 Lecture 6/MBB 222 10

folding

assembly

- initially, the first ~30 amino acids of the polypeptide chain present within the ribosome are constrained and cannot fold until they exit from the ribosome(the amino, or N-terminus emerges first, and the C-terminus emerges last).

- as soon as the first part of the nascent chain is extruded, it will start to fold co-translationally (i.e., acquire secondary structures, domains etc.); as the complete polypeptide is produced and extruded, it will fold in a similar fashion and then the final tertiary structure will be established, followed (in some cases) by assembly of subunits to form the quaternary structure.

Co-translational protein foldingCo-translational protein folding

NH3+

definition: co-translational is a process which occursduring the translation (synthesis) of a protein on the ribosome

September 15, 2003 Lecture 6/MBB 222 11

Molecular chaperonesMolecular chaperones

- the great majority of proteins can fold without assistance, in a co-translational manner

- some proteins, which may have ‘difficulties’ reaching their native states, must be stabilized by molecular chaperones (or chaperonins) by assisted folding

- these bind to nascent (emerging) polypeptides and stabilize them (usually by associating hydrophobic residues).

- otherwise these hydrophobic residues tend to associate with otherhydrophobic residues, leading to intra- or inter-molecularassociations with other proteins that prevent proper folding

- there are dozens of different types of molecular chaperones, and some accomplish functions different from helping protein folding

- e.g., some help protein assembly, some help to transportproteins to various parts of the cell, some help damagedproteins from refolding

September 15, 2003 Lecture 6/MBB 222 12

Most extensively-studied of all chaperonins: the GroEL-GroES complex of E. coli

EL

September 15, 2003 Lecture 6/MBB 222

Protein misfolding can cause serious human diseases e.g. the prion-based, Creutzfeld-Jacob disease (CJD)-basic mechanism for many neurodegenerative diseases is similar the formation of protein aggregates that kill nerve cells.

Prion diseases are self-infectious: misfolded version of the prion protein, PrP*, can induce the normal PrP protein to misfold into a more strand-based structure, resulting in damaging aggregation via formation of cross- filaments.

these filaments are visualized cytologically as amyloid stacks

(see pp362-363 of Alberts et al.)

September 15, 2003 Lecture 6/MBB 222 14

depiction of cross -filament structure resulting from extensive stacking of misformed sheets; this type of structure is resistant to proteases

Model for conversion of PrP tp PrP*: shows the change of two -helices into four strands

September 15, 2003 Lecture 6/MBB 222 15

Protein quaternary structureProtein quaternary structure

Association of multiple polypeptides into a functional unit- many, although not all proteins engage in this- individual proteins in the quaternary structure

are called‘subunits’

Example: prefoldin (a so-called molecular chaperonethat assists the co-translational stabilizationof proteins during their folding in vivo (in the cell)

September 15, 2003 Lecture 6/MBB 222 16

- structure of prefoldin hexamer

- oligomerization (assembly) domain is a double beta-barrel structure composed of beta-strands

- coiled coils consist of two helices winding around each other

two types ofproteins (subunits)

assemble into ahexamer (6 subunits)

Prefoldin quaternary structurePrefoldin quaternary structure

September 15, 2003 Lecture 6/MBB 222 17

Symmetries of ProteinQuaternary StructureSymmetries of ProteinQuaternary Structure

Figure 6.30

Figure 6.32

there are also ways that units can associate into higher order structures without symmetry

September 15, 2003 Lecture 6/MBB 222 18

Protein modifications:requirements for activityProtein modifications:

requirements for activity- cleavage and covalent modifications of proteins (often after synthesis) but may also be co-translational

INSULINSynthesized as PREPROINSULIN- 1st cleavage removes signal sequence (PRE)- 2nd and 3rd cleavages remove joining (PRO)peptide sequences

- di-sulphide bonds hold the two peptidestogether

H2N-

H2N-

H2N-

H2N-

A zymogen is a catalytically inactive protein precursor that must be cleaved proteolytically to be activated

disulfidebonds

I. CLEAVAGEsome proteins require sections of the polypeptide chain

to be removed for correct maturation.

September 15, 2003 Lecture 6/MBB 222 19

- the pancreatic proteases (such as trypsin, chymotrypsin, elastase, and carboxypeptidase) covalent enzyme activation by proteolytic cleavage

- synthesized in the pancreas in an inactive form because if they were active in the pancreas, they would digest the pancreatic tissue. Rather, they are made as slightly longer, catalytically inactive molecules called zymogens (trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase, respectively)

Figure 11.39

*

*

***

*representsactive enzyme

Zymogens in actionZymogens in action

cleavageevent

September 15, 2003 Lecture 6/MBB 222 20Figure 11.40

Activation of Chymotrypsinogen (No Enzymatic Activity)

Chymotrypsin – Serine Protease

One of the most complex examples of proteolytic activation

1st cut is stabilized by S-S bond

chymotrypsin

Series of modifications. Each triggering the next

Final state chymotrypsin

(by Trypsin)(Ile)

Chymotrypsin activationChymotrypsin activation

September 15, 2003 Lecture 6/MBB 222 21

Sometimes proteins are covalently modified after synthesisThese modifications can be:

1. Required to obtain the active conformation (e.g.. collagen)

2. Used to control the activity of a protein (e.g. histones, signal transducing proteins, etc.)

Examples: Collagen: Proline Hydroxyproline (hydroxylation)

This requires Vitamin C; No Vitamin C No Hydroxyproline ScurvyDue to weakening of collagen fibres- hydroxylation of prolines somehow stabilizes structure

N

C R

O

RHN

C OH

O

OH

H

II. Covalent Modifications (p. 403-408)II. Covalent Modifications (p. 403-408)

OH

September 15, 2003 Lecture 6/MBB 222 22

Prothrombin: Glutamate gamma-Carboxy Glutamate (carboxylation)

This requires Vitamin K; No vitamin K No Blood Clotting

H2N CH C

CH2

OH

O

CH2

C

O-

O

H2N CH C

CH2

OH

O

CH

C

O-

OCO

O-

Histones: Histones are proteins involved in the folding/compacting of nuclear DNA. They are often modified in regions of active transcription.

Acetylation of Lysine is the MOST common- (decreases net positive charge of histones).

NH2

CH

C

H2C

OH

O

H2C

H2C

H2C NH2

NH2

CH

C

H2C

OH

O

H2C

H2C

H2C

HN

C

O

Prothrombin, histonesProthrombin, histones

September 15, 2003 Lecture 6/MBB 222 23

Signal Transduction Proteins: Phosphorylation of Hydroxyls (-OH)

- these proteins become transiently phosphorylated which either activates or inhibits their activity

- phosphorylation can be on one of a 3 different amino acids

- a particular protein will only have specific modification sites

Serine phosphoserine

Threonine phosphothreonine

Tyrosine phosphotyrosine

H2N CH C

CH2

OH

O

OH

H2N CH C

CH2

OH

O

O

PO

O-

O-

kinases are the cellular proteins

that phosphorylate these residues

phosphatases are the cellular

proteins that remove the

phosphate groups

together these modulate protein activity

Phosphorylation- an important kind of protein modification.

September 15, 2003 Lecture 6/MBB 222 24

protein activityAsn, Ser, ThrGlycosylation

variousLysC-terminusTyrLys, N-terminusN-terminusCysCysTyrPro, Lys, Asn, AspArg, Lys, His, Glu

Ser, Thr, Tyrtarget site

activationdegradation/otherbioactive peptidesprotein-protein intera’ngene expressionmembrane associationmembrane associationsignalling, oncogenesisprotein-protein intera’ncollagen structureprot. repair, chemotaxis

signalling, activationcellular process

UbiquitylationTruncation

AmidationSulfationAcetylationMyristoylationPalmitoylationPrenylationSulfationHydroxylationMethylation

Phosphorylationmodification

More protein modificationsMore protein modifications