BCHM 3050 Lecture 7.pptx

1

Folding of a 10 Ala Peptide

2

The Anfinsen Experiments

Fig. 5.24

•_{Urea disrupts H-bonds &}

hydrophobic interactions.

•_{-ME reduces disulfides.}

(urea) H₂N–C–NH₂

II O

+

HS–CH2CH2–OH (-mercaptoethanol) Native (active)

Ribonuclease

Denaturation

The disruption (or unfolding) of tertiary &

secondary protein structure that leads to

loss of protein function.

Notes:

Denaturation can be caused by a variety of

treatments and/or reagents.

“Denaturation” also refers to the disruption

of nucleic acid structure & function.

Denaturing Agents &

Treatments

Heat / Temperature

Increases the rate of molecular motion/vibration, causing disruption of H-bonds, leads to protein unfolding.

Strong Acids / Bases

Cause protonation/deprotonation of sidegroups, alters H-bonding & salt bridges.

Proteins become insoluble & precipitate as they reach their isoelectric points.

Water-soluble Organic Solvents (alcohols)

Interfere with hydrophobic interactions; substitute or interfere in H-bonding.

Detergents

Disrupt hydrophobic interactions, leading to protein unfolding.

Denaturing Agents &

Treatments

High Salt Concentration

Interferes with salt bridges / charge interactions in proteins.

Can “extract” water molecules from protein surface, leading to protein aggregation & precipitation.

Reducing Agents

(e.g. -ME, DTT) reduce disulfide bridges; unfolding.

Mechanical Stress

(e.g. stirring, grinding) disrupts weak forces that stabilize tertiary protein structure.

6

Proteins Start Folding As They

Form During Translation

DNA

RNA

Protein

Replication

Transcription Translation

“Information Processing”

Traits

Polyribosomes and

folding nascent peptides. Translation:

The ribosome-mediated biosynthesis of a polypeptide based on the genetic information encoded in mRNA.

7

Energy Landscape Model

for Protein Folding

8

Protein Folding is Assisted by

“

Molecular Chaperones

”

Background on Chaperones

Originally discovered in 1980’s in relation to heat shock stress in higher plants and Drosophila.

Given the descriptive name “Heat Shock Proteins” or “hsp’s”.

Hsp’s conferred thermo-tolerance to organisms subjected to non-lethal high temperature stress.

 Molecular chaperones assist protein folding in two ways:

 Preventing inappropriate protein-protein interactions

 Helping folding occur rapidly and precisely

 Two major classes: Hsp70s and Hsp60s

(chaperonins) Release of polypeptides from hsp70 & hsp60 requires ATP.

 Hsp’s also promote re-folding of proteins

resulting from stress conditions.

 If re-folding is not possible, hsp’s promote

protein degradation

Figure 5.31 Space-Filling Model of the E. Coli

Chaperonin, called the GroES-GroEL Complex

Section 5.3:

Proteins

Section 5.3:

Proteins

Some Diseases Associated with

Mis-folded Proteins

Alzheimer’s Disease

Associated with insoluble fibrous aggregates of 

-amyloid protein (plaques) in brain neurons involved in memory & cognition.

-Amyloid protein is derived from the proteolytic

cleavage of “amyloid precursor protein” (a

transmembrane protein with unknown function).

Proteolytic Processing of Amyloid

Precursor Protein in Alzheimer’s

13

Alzheimer’s:

Aggregation of -amyloid protein to form plaques.

Normal:

Some Diseases Associated with

Mis-folded Proteins

Mad Cow Disease (Bovine Spongiform

Encephalitis)

14

Mad Cow Prions- Proteinaceous

Infectious Particles

17

Normal

Folding Pattern of Pre-prion

Abnormal



Fibrous Proteins



Typically contain high

proportions of

a

-helices

and



-pleated sheets



Often have structural

rather than dynamic

roles and are water

insoluble



Keratin

(

a

-helices)

and

silk fibroin

(



-sheets)

Figure 5.33 a -Keratin

Fibrous

Proteins

Figure 5.34 Molecular Model of Silk Fibroin

Fibrous Proteins



Collagen

:

part of the

connective tissue matrix



Left handed helices twisted to

form a right-handed triple helix



Impart special properties to

structures (e.g., bone and

skin)



Glycine and proline and

hydroxyproline

are common

amino acids

Figure 5.35 Collagen Fibrils

Collagen

Collagen and aging

22

Globular proteins- Structure &

Function

of Myoglobin vs. Hemoglobin

Very similar polypeptide tertiary

structures.

Same heme “prosthetic group” to bind

oxygen.

Very similar functions (oxygen

transport).

Myoglobin is a monomer; Hemoglobin

is a tetramer.

Different physiological locations,

affinities for O

,

kinetics of binding &

23

Functional Relations Between

Myoglobin & Hemoglobin

Hb-O₂

Hb-CO₂ Mb-O₂

Mb

Cells or Storage

MYOGLOBIN

HEMOGLOBIN

• 8 α helices

• Found in skeletal and cardiac muscles • Stores oxygen; has higher affinity for oxygen compared to hemoglobin

• 2 α and 2β helices • Found in RBCs

25

Heme Groups of Globins

101

26

The Hemoglobin

Allsosteric Transition

Oxygen binding causes a 15 rotation of the

a₁₁ dimer relative to the a₂₂ dimer.

Change in conformation increases the affinity / binding of subsequent O₂ molecules.

27

The Hemoglobin

Allsosteric Transition

Oxygen binding causes a 15 rotation of the

a₁₁ dimer relative to the a₂₂ dimer.

28

Factors Affecting

the Amount of 0

Bound by Hemoglobin

1. Availability of oxygen

(i.e. concentration or partial

pressure of O

).

2. Binding of first O

molecule

(i.e. cooperativity)

3. pH

29

Oxygen-Binding Equilibrium Curves

for Myoglobin vs. Hemoglobin

% S a tu ra ti o n

Change in pp O2 causes

changes in O₂ affinity & binding.

 working muscle

30

Effects of pH on O

Binding

H+ Ions decrease hemoglobin’s affinity for O 2.

Sources of H+ ions include bicarbonate buffering system

and lactic acid production.

Both CO₂ and lactic acid accumulate in tissues that are in need of O₂.

Low pH promotes release of O₂; high pH stabilizes HbO₂.

-CO₂ + H₂O HCO₃ + H+

Glucose   Pyruvate   CH₃ – C – COOH O

I

H H

-31

Effects of pH on O

Binding

The Bohr Effect

Low pH (high CO2, high lactic acid) decreases Hb affinity for O2.

H+ ions displace O 2

bound to Hb.

32

Effects of Carbon Monoxide

on O

Binding

CO (carbon monoxide) binds very

tightly to Fe2+ in the heme of

hemoglobin (250 x’s tighter than O₂).

CO-Hb can not carry O₂.

CO binding is cumulative & virtually “permanent”.

CO poisoning leads to asphyxiation. “Mainstream” cigarette smoke has

500 ppm CO.

Cigarette smokers have 5 – 15% CO-Hb.

C III O

33

Effects of CO

34

Other Types of Hemoglobin

Adult (HBA) –

a



configuration

(



-

His143,



-

Glu6)

Fetal (HBF) –

a



(



-143 Ser)

configuration

Sickle Cell (HBS) –

a



(



-6 Val)

35

Sickle Cell Hemoglobin

HBS has Val substituted for Glu at position 6 in  subunits. Position 6 is located at surface of  subunits.

Hydrophobic Val binds to hydrophobic pockets in other 

subunits.

Cross binding polymerizes HBS into sickle shaped

36

Features of Sickle Cell Anemia

HBS fibers distort cells into sickle shapes.

Sickle-shaped cells are more rigid, pass through capillaries with greater

difficulty, impair circulation, cause tissue damage & pain.

Sickle cells are more fragile, targeted by immune system, & turn over faster.

Afflicted people are anemic (i.e. they produce only about half the normal number of blood cells).

HBS is less efficient in carrying O2.

Sickling is promoted in the absence of O₂.

37

Study Questions / Objectives- Lectures 6&7

1. Understand primary structure of proteins

2. Define/describe different secondary structures and

super-secondary structures. Understand how to classify them in a Ramachandran plot. What are turns and loops in the context of secondary structures?

3. Define “tertiary protein structure” and describe how hydrophobic &

electrostatic interactions and hydrogen & covalent bonds can all be involved in stabilizing tertiary protein structure.

4. Describe the nature of the “Anfinsen experiments”, and how or why

they were important in protein folding.

5. What are heat-shock proteins and/or molecular chaperones, and

what is their role in protein folding and turnover.

6. Define “protein denaturation” and describe the main reagents or

treatments that denature proteins.

7. Define “quaternary protein structure” and describe how or why it

38

Study Questions / Objectives

8. How is protein degradation facilitated?

9. Describe prions and how they influence misfolding of proteins causing neurological disorders?

10. Know structures of collagen and its function

11. Describe the structural and functional similarities &

differences between hemoglobin and myoglobin, including the mechanisms of action of any physiological factors that can affect O₂ binding.

12. How is sickle-cell hemoglobin different from normal (adult) hemoglobin, and how do these differences contribute to