WWAMI - MEDS 514 – PROTEINS & ENZYMES TOPIC 3 - PROTEIN FOLDING, UNFOLDING & MISFOLDING

All proteins begin existence as an unfolded linear sequence of aa as they are assembled on a ribosome. Each protein will fold during and/or after protein synthesis until it adopts its native conformation. Each amino acid can exist in different conformations and along the polypeptide backbone there is some freedom of rotation about the N-C and Ca-carbonyl C bonds, the angle of these bonds are defined as Φ (phi) and Ψ (psi), respectively.

Because of steric hindrance, only certain ranges of Φ and Ψ are found in proteins. Ramachandran Plots show the acceptable Φ and Ψ values (Right: calculated for poly-L-Ala) Darkest = no steric interference By definition, Φ & Ψ = 0.0 when the two flanking peptide units are in the same plane.

PROTEIN FOLDING PROBLEM - LEVINTHAL’S PARADOX: When a protein folds, each amino acid can exist in multiple conformations where Φ and Ψ vary.

(1) For simplicity, assume each amino acid can only exist in 10 distinct conformations (10 Φ;Ψ combos).

(2) E. coli can synthesize a complete, folded and active 100 amino acid protein in ~5 sec.

(3) If each aa can adopt 10 different conformations (on its way to folding correctly), then a 100 aa protein is capable of being in 10100 different conformations.

(4) If the 100 aa protein folds completely by random and it takes only 10-13 sec to sample each conformation, how long would it take to sample all possible conformations?

Conclusion: Protein folding can not occur as a completely random process.

How do proteins fold? Certain secondary structures such as -helix and -sheets form rapidly as a result of specific aa sequences & then these secondary structures begin to interact with one another.

Protein Folding Machinery If a protein doesn’t fold or misfolds, this exposes

hydrophobic domains that are recognized by protein folding machinery. Two types: Hsp70/Hsp90 – Heat Shock Proteins

o Use ATP hydrolysis to drive folding Chaperonins

o big machines that use hydrophobic pockets & ATP hydrolysis to drive folding

DENATURATION = unfolding of a protein structure.

Denaturants – unfolding proteins in the lab 1. Increasing temp forces proteins to unfold &

lose native structure and biological activity. 2. Chaotropic denaturants such as urea or

guanidine HCl disrupt H2O H-bonding which disrupts the ‘hydrophobic interactions’ that make up the internal core of globular proteins.

3. Detergents such SDS (sodium Dodecyl sulfate) coat the hydrophobic portions of proteins and give the protein additional charged groups solvated by H2O.

What stabilizes 3D Structure? Hydrogen bonding Ionic bonding Hydrophobic interactions Disulfide bonds

Circular dichroism (α‐helical content) 

Internal fluorescence 

Disulfide Bonds:

Covalent bonds between 2 Cys

Formation requires oxidation; O2 is common oxidant for this

Cleavage requires reduction (DTT & -ME are

common reductants for this purpose)

Cys Disulfides =

SDS PAGE Electrophoresis SDS PAGE sample preparation usually adds the following. What is the function of each?

SDS – Sodium dodecyl (C12) sulfate

-ME - -mercaptoethanol –

Bromophenol blue –

Glycerol – Renaturation of a protein back to the native, folded, active conformation can occur. This is protein-dependent and may also depend on how the denaturant is removed. Christian Anfinsen won the Nobel Prize (1972) denaturing and renaturing ribonuclease. What is the function of ribonuclease?

AMINO ACID SEQUENCE DICTATES 3D STRUCTURE Anfinsen - Expt I Using pure ribonuclease, Anfinsen added: (1) 8 M urea + trace -ME Why urea? Why -ME? (2) Remove urea and -ME slowly

using dialysis. Allow exposure to air (~21% O2).

Dialysis uses a membrane with small holes that allow small molecules to move freely while proteins or other large molecules are trapped on one side. Result: recovered nearly Conclusion: The information needed for proper folding of a protein can exist in the primary amino acid sequence. The primary amino acid sequence dictates the 3-D structure of a protein.

Anfinsen Expt II (1) Denature RNAase with 8 M urea & trace -mercaptoethanol (2) Remove -ME first; keep urea at 8 M. Allow exposure to air. (3) Remove urea second. Result: Why? The disulfides formed There are __ different ways to arrange 4 cystines using 8 cysteines Anfinsen Expt III Using the scrambled, inactive RNAase from Expt II, Anfinsen added a trace amount of -ME & waited 10 hrs. Result: recovered

PROTEINS MISBEHAVING – DEATH BY MISFOLDING

A 58-year old woman, Dolores Mensha was admitted for terminal care with a rapidly progressing neurological disorder including memory loss, headaches, transient episodes of confusion, and blurring of vision. After D. Mensha died (just 7 months after onset), an autopsy of the central nervous system was ordered (as encouraged by the CDC).

Why? Possibility of Transmissible Spongiform Encephalopathy (TSE) TSE can only be verified by:

Data

1. D. Mensha had no history of receiving a corneal transplant or dura matter grafts or human pituitary growth hormone.

So what? Highest infectivity of TSE comes from 2. There was no history of travel outside of the continental US in the past 40 years. So what? Reduces chance of consuming 3. There was no family history of significant neurological disorder. So what? Reduces the chance of 4. No known history of consumption of beef raised outside the US & she did not consume

wild game. So what? reduces chance of consuming BSE-tainted beef or other afflicted animals.

Transmissible spongiform encephalopathies (TSE) are a group of diseases that result from the misfolding of a single protein, PrP, which is also known as a prion.

Prions = proteinaceous infectious particle. PrPSC - infectious prion protein, has a

different 3D conformation than the PrPc

PrPC is naturally found in our neuronal tissues; harmless

PrPC PrPSC

TSE Progression Somehow the infectious form must be able to convert the harmless, noninfectious form into more of the infectious form. Human prion diseases or TSE include: Consumption

kuru – New Guinea

variant Creutzfeldt-Jacob Disease (vCJD) Sporadic or Inherited

sporadic & familial CJD

CJD

Fatal Familial Insomnia (FFI)

Gerstmann-Strassler-Scheinker (GSS) Disease

Trauma

repeated head trauma o football, boxing

Well known animal prion diseases or TSE include:

Bovine spongiform encephalopathy (BSE)

Chronic Wasting Disease

Scrapie

Feline spongiform encephalopathy (FSE)

~20 Mutations in the human PrP gene known to favor disease

Codon 129 has gained particular prominence because, in all but 1 or 2 known cases of vCJD, whether transmitted by consumption of infected beef or through medical procedures, codon 129 is homozygous for Met!

In the UK general population: M/M occurs 42%, M/V occurs 47%, V/V occurs 11% Does this mean if you are homozygous for Met-129 that you won’t get CJD from BSE? You may not get vCJD but some have warned that any disease requiring long incubations might not have surfaced yet.

Key features of the prion diseases (1) PrPSc is resistant to degradation by proteases Indeed, proteinase k digestion of human CJD brain tissue produces a stable 27-30 kD fragment of PrP that is diagnosed by SDS PAGE & Western blots. (2) PrPSc is able to recruit the host PrPC and convert it to the PrPSc form. (3) PrPSc forms insoluble plaques that ultimately destroy cells & generate a

sponge-like appearance within the neuronal tissue. (4) Different PrPSc are capable of forming different, stable 3D conformers which ultimately generate different symptoms/diseases. The autopsy of D. Mensha: (1) histopathology revealed wide-spread spogiosis of the brain but no amyloid plaques (2) SDS-PAGE & Western blot analysis revealed a partial PrP protein band between 27-30 kD (3) Analysis of the cerebrospinal fluid revealed elevated 14-3-3 protein (consistent with CJD) (4) Sequencing of the PrP gene revealed codon 129 was homozygous for Met. 90% of all CJD

patients are homozygous for either Met (fast progressing CJD) or Val (slower progressing).

Elevated levels of 14-3-3 are not usually seen in vCJD patients. Combined with D. Mensha’s history, it looks like D. suffered from classic CJD, not vCJD. There are other tests, including EEGs which are used to distinguish between the various TSE.

Fatal familial insomnia (FFI), for example, results from targeted PRPSc plaque formation in the thalamus, an area of the brain that mediates sleep. (Read “The Family That Could Not Sleep” DT Max)

Neurodegenerative Amyloidoses: Alzheimer’s, Parkinson’s, & Huntington’s: All late-onset neurodegenerative disorders are caused by accumulation of:

Amyloid plaques: named by analogy to plant starch (amyloid) deposits Neurofibrillary tangles (NFTs): different proteins

1. Alzheimer’s Disease Extracellular amyloid deposits of proteolytic fragments of

the amyloid precursor protein (APP) termed

Intracellular NFTs composed of the

Aβ = peptide of ~40 amino acids; Polymerizes to form long

insoluble fibrils consisting of H-bonded parallel β-sheet structure.

Why does Aβ aggregate in some people and not others?

2. Parkinson’s Disease: Accumulation of neurofibrillary tangles (NTFs) composed

of polymers of α-synuclein protein. No evidence of amyloid plaques

Tau and α-synuclein have been shown to interact and many Parkinson’s patients have symptoms of Alzheimer’s and vice versa. Mutations in genes other than SCNA (coding for α-synuclein) are also associated.

3. Huntington’s Disease: 1872 : Autosomal dominant attributed (1993) to Nuclear protein deposits are polyglutamine (polyQ)

aggregates & form β-sheet enriched amyloid-like fibrils. Aggregates arise from mutant forms of huntingtin, a

protein with a long stretch of NH2-terminal Gln. Huntingtin: 3144-aa protein encoded by 67 exons – exon 1

contains a CAG repeat region coding for polyglutamine. Most people have < 27 CAG (codon for gln) repeats & never develop the disease. How many CAG repeats guarantee disease within a normal lifetime?

Genetic screening is

SUMMARY - TOPIC 3 - PROTEIN FOLDING, UNFOLDING & MISFOLDING

Proteins fold during and/or after protein synthesis until it adopts its native conformation. Some proteins require folding assistance from chaperone (Hsp70, Hsp90, etc) machinery.

There is some freedom of rotation about the N-C and C-carbonyl C bonds, the angle of these bonds are defined as Φ (phi) and Ψ (psi), respectively.

Levinthal’s Paradox: If a protein folded completely by random, it would take much too long to sample all possible conformations (>1077 yrs for 100 aa). Therefore, folding can not occur by completely random processes.

The 3D structure of proteins is stabilized by a combination of hydrogen bonding, ionic bonding, van der Waals forces, hydrophobic interactions & disulfide bonds.

Denaturation: unfolding of a protein structure. Increasing temperature, for example, forces proteins to unfold as they lose both their native structure & biological activity.

Chaotropic denaturants (urea, guanidine HCl) unfold proteins by extensive disruption of H2O H-bonding to the extent that the hydrophobic portions of the protein can open up and be exposed to the solvent. Detergents like SDS coat hydrophobic regions of proteins which causes them to unfold.

Cystines describe the covalent disulfide bonds occurring between 2 Cys. Formation of the disulfide requires oxidation (O2 is often the culprit). Disulfide cleavage requires reduction; common lab reductants include dithithreitol (DTT) and -mercaptoethanol (-ME).

Christian Anfinsen denatured and renatured ribonuclease (RNAase) and found that all the information necessary for the protein to adopt its native, active conformation was present in the aa sequence (i.e. AMINO ACID SEQUENCE DICTATES 3D STRUCTURE).

Anfinsen’s 1st expt: (1) unfolded RNAase with 8 M urea and broke disulfides with -ME; (2) removed both urea & -ME slowly (dialysis) & allowed exposure to 21% O2. Result: recovered ~100% activity; the protein folded back into its native, active conformation.

Anfinsen’s 2nd expt: (1) unfolded RNAase with 8 M urea and broke disulfides with -ME; (2) removed -ME first & exposed to air (21% O2); (3) removed urea. Result: ~1-2% activity; the disulfides reformed randomly in the presence of the urea.

Anfinsen’s 3rd expt: (1) using the scrambled protein from expt 3, add -ME. Result: after 10 hr, he recovered ~100% activity. Amino acid sequence specifies conformation.

The transmissible spongiform encephalopathies (TSE) are caused by misfolded forms of the prion protein, PrP. The misfolded form of the protein, PrPSC, is especially resistant to protease treatment (& heat treatment) which allows the protein to persist in the neuronal tissues. In addition, PrPSC is capable of converting the normal, benign form of the prion protein, PrPC, into more of the PrPSC, which accounts for the accumulation of the protein. The accumulations of PrPSC form amyloid-type plaques which ultimately perforate cells causing a spongiform destruction of the brain. Different forms of the disease-causing PrPSC preferentially affect different regions of the brain. Fatal Familial Insomnia (FFI) patients have preferential plaque formation within the thalamus which dramatically affects their ability to regulate sleep; patients with this particularly cruel disease can spend their last few months with little or no sleep.

With regard to TSE, the sequence of the PrP protein matters. There are certain sequences that favor one or more forms of TSE. The mad-cow form of the disease, vCJD, has a strong bias for Met at codon 129. Close to 200 people developed the variant form of CJD, vCJD, after eating mad cow tissue. Every single one of them was homozygous for Met (M/M); only 42% of the general UK population is homozygotic (M/M).

All late-onset neurodegenerative disorders are caused by accumulation of protein aggregates or fibers. These usually take the form of amyloid plaques or neurofibrillary tangles (NFTs).

Alzheimer’s Disease: Extracellular amyloid deposits made of proteolytic fragments (40 aa membrane spanning peptide) of the amyloid precursor protein (APP) termed amyloid-β (Aβ); is for the -strands formed by the 40 aa. Also: intracellular NFTs composed of the microtubule-binding protein tau ().

Parkinson’s Disease: Accumulation of neurofibrillary tangles (NTFs) composed of polymers of α-synuclein protein; no plaques. α-synuclein & tau interact; some Parkinson’s patients show symptoms of Alzheimer’s and vice versa.

Huntington’s Disease: protein deposits originate as nuclear inclusions composed of polyglutamine (polyQ) aggregates & form β-sheet containing amyloid-like fibrils. The polyQ appears on the 3,144 aa huntingtin protein; exon 1 contains a CAG repeat that encodes Gln. Most people have < 27 CAG (codon for Gln) repeats & never develop the disease. ≥40 CAG repeats develop the disease within a normal lifetime. Genetic screening is definitive.