Biochemistry 5012: Section on Protein Biochemistry

Lecturer: Chuck Sanders

Lectures 7-8

Protein Stability and Folding Protein Folding and Disease

Learning Objectives To understand the basic principles and diversity of protein stability.

To understand the energetic basis for rates of processes: kinetics.

To understand the nature of protein folding.

To survey the relationship of protein folding and human disease.

Topic Outline Thermodynamic stability of proteins Stabilizing forces for folded proteins Kinetic and cellular stability of proteins Introduction to kinetics Free energy barriers and rate limiting steps The nature of protein folding pathways Mutations and perturbation of protein folding and stability Protein denaturing agents Protein stabilizing agents Protein folding under cellular conditions Chaperones Folding Quality control Protein folding and disease CFTR and cystic fibrosis PMP22 and Charcot-Marie-Tooth Disease Rhodopsin and Retinitis Pigmentosa Complex disease and non-genetic factors contributing to misassembly

7000 6000 5000 4000

Human Gene Mutation Database (Cardiff)

www.hgmd.org

Recall, there are now 53,000 known disease linked inherited mutations, about 50% of which lead to a single amino acid changes in a protein.

Based on preliminary analysis of how some of these mutations perturb the affected protein, it appears that problems with protein folding may be the most common class of molecular defect that leads directly to human disease.

3000 2000 1000 1986 1988 1990 1992 1994

Year

1996 1998 2000 2002

The unfolded form is not a single conformation, but composed of dynamically interconverting conformations.

Thermodynamic Stability of Proteins

unfolded folded K

eq,fold C N

K =

eq,fold [folded] [unfolded]



G

o

Standard Free Energies of Folding for Single Domain Proteins Protein



G o fold (kcal/mol)

lambda repressor alpha spectrin SH3 domain arc repressor cytochrome C (with heme and Fe(II) CD2 procarboxypeptidase U1A spliceosomal protein Hpr -3.0

-2.9

-6.3

-15 -8.2

-4.1

-9.3

-4.6

Proteins are only moderately stable:

The folded state is usually favored over the unfolded state by only -3 to -10 kcal/mol.

Where Does the Energy Come From to Stabilize Proteins?

We have already discussed hydrogen bonding, the hydrophobic effect, and so forth – all of these make contributions. One additional interaction that should be introduced is disulfide bond formation.

unfolded folded S S



G o no disulfide bond K eq,fold C N S S Disulfide bonding shifts equilibrium towards the folded state by destabilizing the unfolded state

.



G o with disulfide bond

energy by which disulfide bond formation destabilizes the unfolded state by lowering its conformational entropy

For Globular Water Soluble Proteins:

Except for functionally-related pockets (such as active sites), protein interiors are generally hydrophobic, with hydrogen bonding potential of backbone and (relative rare) polar side chains being satisfied by protein-protein H-bonding. This is very efficiently accomplished by having interiors dominated by β-sheets and



-helices.

Protein exteriors are polar, with extensive water/polar side chain interactions. Loops and turns are mostly on the surface, where there is plenty of water available to satisfy the H-bonding potential of the backbone amides.

Balance Sheet for Protein Folding: Ribonuclease T1

Adapted from C.N. Pace et. al. FASEB J. 10, 75-83 (1996). Ballpark estimates, but qualitatively correct.

Stabilizing: Destabilizing: salt bridges: -10 kcal/mol loss of conformational entropy: 177 kcal/mol (1.7 kcal per 104 residues) disulfide bonds: -7 kcal/mol burial of about 150 hydrophobic carbons: -112 kcal/mol Transfer of 73 peptide groups from full exposure to water to protein interior: 81 kcal/mol replacement of 104 protein-water hydrogen bonds with protein-protein and water-water H-bonds 1(1/7 kcal/mol each): -166 kcal mol burial of polar groups: 28 kcal/mol Net: 286 - 295 = -9 kcal/mol in favor of folding

While protein folding is stabilized by hundreds of contributing interactions, each of which is energetically significant, these are off-set by a large number of destabilizing interactions: the NET energy difference in favor of folding is quite modest.

The term “protein stability” is used in different contexts to describe different phenomena. In addition to thermodynamic stability there are:

Kinetic Stability (a.k.a. Thermal Stability)

“irreversible” aggregation

An “irreversible” process is leads to a state from which there is no return on the relevant time scale (because it is either highly favorable from a thermodynamic standpoint and/or because the barriers to other possible states are so high)

Cellular Stability

Proteolyic Degradation

Both class of stability are usually described by their half-lives (time 1/2 ) for complete aggregation or degradation.

Typical t 1/2 for protein cellular stability: hours to days. Sometimes much longer or shorter.

Protein Folding Pathways: Introduction to Kinetics



G o Thermodynamics provides the difference in energies between states and inidicates whether “products” (i.e., folded state) or reactants (i.e., unfolded state) are favored. However, it tells nothing about the energetics of the pathway which is taken to get from one state to another. This is very important because the nature of the available pathway(s) will determine the rate of conversion between states. Indeed, if the pathways are “difficult”, the process will occur at a negligible rate, no matter how favorable the



G o .

Just because of process is thermodynamically favorable, doesn’t mean the process will occur promptly – thankfully! Consider the factor that peptide bond hydrolysis by water is extremely favorable from a thermodynamic standpoint… and yet peptide bonds spontaneously hydrolyze only very slowly.

Consider the simplest two state process, for which there is only a single energy barrier between states.

2 states 1 barrier in each direction



G * forward



G * reverse 2 states 2 barriers in each direction 1 intermediate

Some processes have more than one rate barrer, in which case the barriers are separated by intermidate states. If a single barrier for one direction of the reaction is much larger than the others, it is referred to as the “rate limiting step” of the process.



G o



*

rate is proportional to rate constant, k, which is proportional to: 

G *

e

RT

There is an inverse logrithmic relationship between rate and activation energy.

k = A .

e



G RT * (the Arrhenius equation)



G o

Intermediate rate limiting step to forward reaction

G* ln k = + ln A (linear form of Ar. eq.) RT Rate is directly proportional to rate constant: k R is the gas constant, T is the absolute temperature, and A is a constant which is characteristic of a given process (but usually not interpreted.)

Relationship of Protein Stability to Folding Kinetics

unfolded folded

C k

fold

k

unfold

N [folded]

K = =

eq,fold

[unfolded]



o

G = -RTlnK

eq,fold

k

fold

k

unfold

k are rate constants

Think of pathways between states in terms of Bathsheba Bear and a honey tree in the valley on the other side of the mountain… Bathsheba can get there from more than one starting point (true for unfolded proteins). However, there is but one destination (true for folding).

BB can take more than one path, but will usually take the easiest path (except on days where BB is feeling especially bright-eyed and bushy-tailed. If more than one equally easy path is available, BB will have little preference for one easy path relative to the others (same with protein folding).

The difficulty of the easiest pathway(s) is what will determine how often Bathsheba Bear makes the trip – BB’s “rate limiting step” (same with protein folding – rate is limited by the highest obligatory barrier).

In the country where BB lives, there are a number of abandoned coal mine shafts that are very dangerous. BB’s ancestors have constructed barriers to ensure that ursi do not fall into them. Nevertheless. tragic accidents sometimes do happen (when this occurs in the protein world, the consequences of trapped and incorrectly folded protein can be equally tragic).

There are preferred folding pathways

“Leventhal’s paradox”: Consider a protein of n residues • there are 2 torsion angles ( y & f) for each residue that must be adjusted • assume there are three energetically favorable (possible) conformations for each residue – therefore, there are 3 2n ≈ 10 n possible conformations • assume that each torsion angle can reorient really fast: with a frequency of 10 13 s -1 This leads to an estimate that the time required for a 100 residue protein to systematically explore all possible conformations is: time = 10 100 /10 13 s -1 = 10 87 s Since the age of the universe is thought to be “only” 10 18 to the conclusion: s, this leads

Proteins do not fold by a random search of conformation space – there are preferred pathways.

The “funnel concept” of hyperdimensional protein folding pathways. Because it is hard to conceptualize multiple dimensions, it is customary to persist in describing the energetics of protein folding using standard “single pathway” concepts and energy diagrams, while not forgetting that reality is more complex.

Protein Folding Pathways

Usual order of folding events

•Secondary structures formed quickly (local)

(

helix formation in µs) •Secondary structures form motifs •Hydrophobic collapse of motifs to form rough domain structure (but remains molten globule-like) •Final fold of domain is locked in.

•Overall time for folding mses-sec.

Mutations can perturb the heights of the rate barriers and/or the energies of the free energy states. Consider a mutation that lowers the energy of a folding intermediate:  unfolded

G o Mutation that lowers energy of folding intermediate (only).

Examples of Potentially Deadly Mutations Barrier to folding becomes too high: protein folds too slowly and is degraded OR heads down an alternate pathway which leads to irreversible misfolding.

Normal barrier to misfolding is reduced so that alternate pathways leading to the misfolded form become efficient.

folded

Folding intermediate is stabilized. giving intermediate time to irreversible aggregate before completing folding.



G o Note that this mutation not only makes the intermediate much more stable (longer-lived), but that the energy barriers from this state to both initial and final states are now larger than for wild type.

Folded form is destabilized, shifting equilibrium towards unfolded form, increasing the time spent in that aggregation-vulnerable form.

Perturbation of the interactions of a nascent protein with the cellular protein folding machinery, so that assistance normally received is absent or corrupted to a bad end.

Protein “denaturing agents” reversibly unfold proteins. Proteins will also “melt” at a critical temperature.

Denaturation = unfolding of a protein.

Chaotropic agents such as urea and guanidinium can reversibly unfold many proteins – they shift the folding equilibrium to favor the unfolded state.

Sodium dodecylsulfate, SDS, is also a denaturing agent. It is a little unusual because it promotes a specific unfolded structural state in which SDS forms rod shaped complexes with the denatured protein. SDS is a detergent.

Unfolding of a protein also occurs when a critical temperature is exceeded at which there is a dramatic change in the folding equilibrium constant to favor the unfolded state.

Lambrechts et. al.

Biochemistry 2002 3:12 J. Biochem. 2001

Denaturation shifting equilibrium to favor the unfolded state of the protein.

Denaturation is not the same as aggregation, where protein chains stick together and sometimes precipitate.

Denatured protein tend to be very susceptible to aggregation unless a denaturing agent is present.

Thermally-induced unfolding is often a step en route to irreversible aggregation.

Protein Stabilization Agents these include high concentrations of neutral or zwitterionic osmolytes such as: glycerol trimethylamine oxide betaine

Ligand binding also usually stabilizes proteins – for some proteins nanomolar concentrations are effective. This fact is the basis for an emerging paradigm in rational therapeutics – a way of rescuing a protein whose stability has been compromised as a results of mutation or some other factor.

Unfolded protein

K

1

unfolded

Folded Protein + Cognate Ligand Protein-Ligand Complex

K

2

K

overall

= K

1

x K

2



G o

folded folded protein-ligand complex

Protein Folding In The Context of the Cell In vivo: in a living cell In vitro: usually means cell has been lysed (popped open) but that cellular components are still present. In common usage, it is also used (inappropriately) to describe studies that are carried out under “test tube” conditions using purified molecules…

From: Mathews

From: Alberts

In lower organisms there is a single copy of each gene.

In diploid organisms, there are two copies (“alleles”) of most genes.

The main locations of protein folding in the cell are the cytosol and the endoplasmic reticulum.

Peroxisomes and mitochondria are also sites for for protein folding (of their cognate set of proteins). Proteins are delivered to other organelles in pre-assembled form. There are two general routes for protein transport: (1) diffusion ; this may be random (Brownian motion) or directed (for example, getting carried along microtubules) and may involve gates between compartments (e.g., the nuclear pore that serves as the portal between the cytosol and the nucleus.) (2) vesicular trafficking : some proteins are transported by being trapped in, or surface associated with transport vesicles that bud off from one organelle, diffuse to another organelle and fuse with the membrane of that organelle. Vesicles are bounded by a lipid bilayer.

Alberts

Protein folding in the cell…

While folded structure is determined by primary sequence and thermodynamics (as under test tube conditions), folding pathways may be different in cell than in test tube with pure protein for the following reasons: Folding is often co-translational. This means that N-terminus starts folding as is comes out of the ribosome, even as the C-terminus is being synthesized. This is especially important for multi-domain proteins because it allows domains to fold in an orderly sequential (one by one) fashion.

Chaperone proteins often play important roles in assisting folding and suppressing misfolding/aggregation in the cell.

Besides chaperones, there are a variety of other folding accessory proteins.

Folding is monitored by protein folding quality control.

Protein that is judged to be hopelessly defective by quality control is degraded (hopefully!).

Protein Chaperones

• • • • •

Chaperones are proteins that recognize and selectively bind incompletely folded proteins to form relatively stable complexes.

Chaperones prevent irreversible aggregation of nonnative conformations and keep proteins on the productive folding pathway.

Some maintain newly synthesized proteins in an unfolded conformation suitable for translocation across membranes.

Chaperones are catalysts in the sense that they undergo reversible interactions with their substrate proteins, such that a single chaperone can catalyze folding of many protein molecules through repeated action.

Some chaperones consume ATP or other forms of energy to enforce conformational changes on their client proteins. Other act in an energetically-passive manner.

Proteins That Assist Protein Folding

Note: all of the following might loosely be referred to as “chaperones”

Chaperonins: include GroEL/GroES

chaperonins consume ATP, engulf proteins in folding chamber before release

Chaperones: include DnaJ/DnaK

usually consume ATP, thought to form complex with protein

Small Heat Shock Proteins : include



crystallins,

usually do not consume ATP, usually binds to local structural motifs

Calnexin/Calreticulin: endoplasmic reticulum-specific lectin chaperones, do not consume ATP Petidyl Proline Isomerase (PPIase): catalyzes transition between cis and trans Pro Protein Disulfide Isomerase (PDI): catalyzes disulfide exchange to help protein find right combination of disulfide bonds A number of specific proteases: Some proteins do not fold until proteolytically processed. Other proteins have an internal “chaperone” domain which gets clipped off after the protein folds properly.

… And many others

Modes of Action of Chaperones (Top) and Chaperonins (Bottom)

Ellis and Hartl, Curr. Opin. Struct. Biol. 9, 102-110 (1999)

Protein Folding Quality Control Systems

A system for most secreted and membrane proteins.

A system for cytoplasmic proteins.

These systems are linked to each other in multiple ways.

Proteins in the cell are usually degraded either by being polyubiquitinated and then chopped up in proteasomes or by getting trafficked to the lumen of the lysosomes (where proteins are destabilized by low pH and then degraded by lysosomal proteases).

The Cytosolic Polyubiquitination/ Proteasomal System

Figure by C. Gordon, MRC

ubiquitin (Ubi): a small protein that serves as a tag to mark proteins for degradation E1: ubiquitin-activating enzyme (ubiquitin is activated by covalent attachment to E1) E2: ubiquitin-conjugating enzymes (receives ubiquitin from E1and then transfers it to targeted protein) E3: ubiquitin ligases: works with E2 to catalyze covalent transfer of ubiquitin to target protein Proteasome: recognizes polyubiquitinated proteins, engulfs them, and hydrolyzes them

Protein Folding Quality Control In the Secretory Pathway

from Alberts

Relevant Proteins:

•

all proteins that are secreted from the cell (including components of extracellular matrix)

•

all membrane proteins destined for the cell surface

•

all soluble and membrane proteins destined for ER, Golgi, endosomes, or lysosomes

Other Facts:

•

the ER often takes up >10% of the cellular volume

•

probably 25-40% of all proteins either reside in or pass through the secretory pathway

•

most folding quality control of the secretory pathway is localized in the ER

Calnexin Cycle Slides

A key component of ER quality control is the calreticulin/calnexin cycle. This cycle appears to involve the interplay between the lectin chaperones (calnexin and calreticulum), a folding sensor (UDP-glucose glycoprotein glycosyltransferase), and a mannosidase that serves as a degradation timer.

Protein that fails to fold correctly gets targeted for degradation by the “ERAD” (ER-associated protein degradation) system. This involves retrotranslocation out of the ER via the translocon pore, polyubiquitination, and degradation by proteasomes.

When quality control and/or ERAD gets overwhelmed, activation of the “UPR” (unfolded protein response) is triggered.

The “unfolded protein response” involves signaling from the ER to the nucleus that leads to the activation of transcription of dozens of genes. Many of these genes are stress response proteins, such as chaperones. One outcome of UPR action is an expansion of the size of the ER to accommodate the excess protein accumulating there.

(David Ron, J. Clin. Invest., 2002) no need to memorize details

Back to Protein Folding

and Disease

…

Protein Biogenesis is not Always Efficient

Ribosome Folded Protein Nascent Protein Misassembled Protein

loss of function

and/or

possible toxicity

(a class of “gain of function”)

Contributions of Protein Misassembly to Human Disease

Simple Disease: Defects in a single gene lead to disease. Usually in the form of heritable mutations.

Complex Disease: Defects in a single gene or protein, either inherited or somatic are a risk factor. Other factors besides mutations may also trigger protein misassembly.

Sporadic/Acquired Disorders: A disease that arises due to non inherited events, such as somatic mutations. May be “simple” or may be “complex”.

Infectious Disease: May be triggered or aggravated by defects in genes and/or protein.

Selected Simple Heritable Disorders Known to Involve Protein Misassembly

Cystic Fibrosis Charcot-Marie-Tooth Retinitis Pigmentosa Some Cardiac Arrhythmias CFTR PMP22 Connexin-32 Rhodopsin Rom-1 Peripherin Potassium Channels Diabetes Insipidus Familial Alzheimer Disease Aquaporin-2 Vasopressin Receptor Beta Amyloid Precursor Presenilin

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR):

An epithelia chloride ion channel. Key function is to regulate salt in epithelial cells – with lung airway cells being especially relevant to CF.

Cystic Fibrosis:

A recessive disorder (both copies of gene must be defective). Afflicts 1:3000.

Most patients die of lung infections. Average life span of patients has increased dramatically over the years.

Figure: Lukacs and Durie New Eng. J. Medicine, 2003

Hundreds of different single site point mutations in the CFTR chloride channel can cause cystic fibrosis. However, the majority of patients are afflicted by the



F508 mutation. This mutation results in CFTR never trafficking beyond the ER – the defective form of the protein is then degraded by the proteasome. Failure of this protein to fold and traffic beyond the ER leads to loss of its chloride channel function on the surface of airway epithelial cells. Remarkably, when this



F508 mutant form is artificially coaxed to fold and traffic to the membrane surface, it is observed to function.

F508 is located in NBD-1 and likely helps to organize NBD-1 with respect to the transmembrane domain

Peripheral Myelin Protein 22 and Charcot-Marie-Tooth Disease

Electron micrograph of myelin.

Myelin Sheath Surrounding A Peripheral Nervous System Axon, With Some of its Key Proteins.

www.hosppract.com

Peripheral Myelin Protein 22

In vivo efficiency of folding of wild type PMP22 is ca. 20%.

The efficiency of some disease-related mutants is near 0.

Myelin

L W Q D

Membrane

T A N C S T 41 H G S N G S S S I V W Q S 26 I V T L F L V V V A V H L V I I L L L L S L L 18 11 Q S G N V W L E 60 N P S H S H C S V Q M A T S I L I I 76 Q S F F I S L L F L L F F C 85 68 T F R L T K G G R 93 H E P V Y T A A M V L G I Q I 100 G C L F Y I S I F T 116 A A W 122 H L Y S 132 Y N D S G F A Y P V I W V L L V I L L A A A S I Y G F L R 143 150 K R E -COOH 160 Sites for which point mutations lead to Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth Disease Involves Defective Myelination in the PNS Normal Myelin: Defective Myelin Hypomyelination Onion Bulbs Onion Bulbs

Allan Burr Steinbach, UC Berkeley “

Disorders of Myelination” Web Site

Club Foot: Occurs in Severe Phenotypes of CMTD

Charcot-Marie-Tooth Disease Type 1A (CMTD1A)

Most common cause of the disease is a third allele of the PMP22 gene – results in too much PMP22 protein.

More rare forms of the disease are caused by dominant mutations in a single PMP22 allele. In other words, even though a patient has one good copy (allele) of the PMP22 gene, having one bad copy is enough to cause the disease.

Mutant forms of PMP22 have been documented to misfold in the endoplasmic reticulum, where they are dealt with by ER quality control and targeted for degradation.

Disease symptoms are worse for the WT/mutant case than for WT/null (where the second allele is missing altogether). How can it be worse for the second allele to be

defective than for it to be completely absent?

This question is answered by the fact that PMP22 dimerizes in the ER. While the mutant forms of the PMP22 are folding-defective, they can still dimerize with the wild type PMP22 in the ER, so that it drags the wild type protein down with it (when it gets targeted by quality control for degradation)!

While there is almost certainly a “loss of function” component to CMTD1A, it is hard to understand the fact that symptoms are only observed later in life (even though it is an inherited disease). This may reflect the fact that as we age our quality control systems weaken – if defective PMP22 is not disposed of properly later in life, the misfolded protein may be toxic — this may complicate loss of function and trigger the onset of the disease symptoms.

Peripheral Myelin Protein 22

In vivo efficiency of folding of wild type PMP22 is ca. 20%.

The efficiency of some disease-related mutants is near 0.

Myelin

L W Q D

Membrane

T A N C S T 41 H S G W N I V Q G S S S 26 W L E 60 N I V T L F L V V V A V H I I L L L V L S L L L 18 11 Q S S G N V P S H H C T S S V Q M A T S I L 76 I Q I S F F I S L L F L L F F C 85 68 F R H P 122 Y T A A M V L G I V Q S C L I I F 100 F G Y I T 116 A A R L T K G G 93 E W H L Y N D S S Y 132 G V I F A V Y I W V P L L L L A A A S I Y G F L R 143 150 K R E -COOH 160 Sites for which point mutations lead to Charcot-Marie-Tooth Disease Documented CMTD-1A Mutations His12Gln Leu16Pro Val30Met Asp37Val Ala67Pro Met69Lys Ser72Leu Ser72Pro Ser72Trp Ser76Ile Ser79Cys Ser79Pro Leu80Pro Gly93Arg Gly100Arg Gly100Glu Leu105Arg Gly107Val Cys109Arg Thr118Met Leu147Arg Ser149Arg Gly150Asp Gly150Cys Arg157Gly Arg157Trp

Rhodopsin and Retinitis Pigmentosa Rhodopsin is the primary rod cell photoreceptor of the human retina.

39 L M Y N G T>K E G P Rhodopsin is a G protein-coupled receptor that is activated when a photon is absorbed by its retinal (vitamin A-derived) cofactor. The activated receptor triggers intracellular signaling though interactions with G proteins.

10 Mutations in rhodopsin result in retinitis pigmentosa – N F Y V P F S the most common cause of blindness among the non-elderly. 85% of disease mutations lead to E F P>H,A ,L S N>S Y Y R V 20 V G T >M A 180 misassembly of the protein.

280 S N S>P E>K F G 200 79 100 119 L >P M> R,K M>R 211 221 260 240 P I F M T> P 290 I F P A>E F 300 S>R A A K>N ,E,M A Y N V P I I Y M F M N 310 K Q N R L C T M T I 320 C V S T K T 340 A S A E E T>M S C G K N D D 330 G L A P> A,R,Q,L,S,T 348 -COOH Q V>L ,M A> P P

Example of a Rhodopsin Mutations Which Lead to Retinitis Pigmentosa Pro267Arg Pro267 site in Rhodopsin RHOD = rhodopsin BOPS = blue opsin VSPR = vasopressin receptor FSHR = follicle stimulating hormone receptor TSHR = thyroid stimulating hormone receptor LHCR = leuteinizing hormone receptor MC4R = melanocortin 4 receptor MC2R = melanocortin 2 receptor ETBR = endothelin B receptor GRHR = gonadotropin-releasing hormone receptor Pro303 site in Rhodopsin retinitis pigmentosa tritanopia diabetes insipidus amenorrhea various thyroid disorders sexual development disorders obesity glucocorticoid deficiency Hirschsprung disease hypogonadotropic hypogonadism highlighted sites in the above sequences: mutation at these sites leaks to disease

Example of a Rhodopsin Mutation That Leads to Retinitis Pigmentosa Pro267Arg : Results in loss of a proline that is required for inducing a kink in a transmembrane helix. This is compounded by introduction of highly charged residue into the apolar membrane interior. Pro267 is conserved in structurally-related G protein-coupled receptors. Mutation of this site in some of these receptors results in other diseases. The Pro267Arg form of rhodopsin never makes it to the rod outer cell membrane of the retina where this protein is required for vision.

Pro267 site in Rhodopsin Pro303 site in Rhodopsin Pro303 in rhodopsin: no disease-linked mutations have been documented (yet). However, this proline is conserved in related receptors. In some of them mutations in this proline is linked to disease. While 3-D structures of those receptors have not been determined, they will resemble rhodopsin (for which structure is known). In rhodopsin Pro303 interrupts the 7 th transmembrane helix. It is easy to see how changing such an unusual and highly conserved site could have catastrophic consequences!

Examples of Complex or Sporadic Disorders for Which Protein Misassembly can be a Factor Type II Diabetes Cancer Common Forms of Alzheimer’s Disease Atherosclerosis amyloid formation by the islet amyloid peptide (amylin) mutations in the p53 tumor suppressor protein (although not the most cancer-linked mutations) common variants of Apolipoprotein E ApoE does not itself misfold. Rather, the disease-linked variants appear to interact differently with the amyloid-beta peptide, in a way that makes it more amyloidogenic.

rare mutations in the low density lipoprotein receptor (resulting in hypercholesterolemia)

Protein misassembly may be triggered by gene mutations, but even wild type protein can misassemble as a result of any of the following factors:

chemical modification (possibly enzyme-mediated) heat (as in fever) cold (as in frostbite) oxidative stress toxic agents (including drugs and smoking) defective protein trafficking over- or underexpression of protein kidney dialysis medical implants defective protein folding quality control system inflammation burns