PROTEINFOLDING

Concepts&Stat.Mech.Models(ofK.Dill)

DNA

T
G
AC

transcription

Butfirstsomemotivations:

splicing
RNA

RNAi

A
C
UG

translation

HumanGenomeDNAis
almostknowninitsentirety.
Genomehas~20,000genesthat
encodeforevenmoreproteins

proteinfolding?

functionalunfolding??

functional,interacting
structures

Avidin#2
Avidin#1

Youcanprovetoyourself
thatthegivenstructureis
adimeroftwoproteins
byopeningthePDBfile
inMSWordandjust
deletingtheBchain.
Thefileformatisshown
onthenextpage.

Hydrophobicbridge
ofassociation

NotethattheapproximatedimensionsofthehydrophobicbridgehasroughlyacrosssectionalareaofA~1nmx1nm(betterseen
withBall&StickDisplay).Suchanareacorrespondstoanassociationenergyof~2* *A.Taking tobe40mN/m,typicalofoil
waterinterfaces,thisassociationhasanenergyof~20kBTbiol.

Figure 4-30 Essential Cell Biology ( Garland Science 2010)

Figure 4-23 Essential Cell Biology ( Garland Science 2010)

Figure 3-23 Essential Cell Biology ( Garland Science 2010)

Figure 3-22 Essential Cell Biology ( Garland Science 2010)

[A] = Atot - [

[AB] / K = (Atot
Figure 3-19 Essential Cell Biology ( Garland Science 2010)

[AB]/Atot

FractionalsaturationofAtot

[AB]/Atot=[B]/{(1/K)+[B]}

1
bo
per
y
h

la

=
(1

[B]
/K
)

1kcal/mol=4.2kJ/mol=1.7kBT

Figure 3-20 Essential Cell Biology ( Garland Science 2010)

CD47 claimed to function as a Marker of Self

flow

SIRP

C
D46

CD47

membrane

Hatherley, Graham, Turner, Harlos, Stua

Prototypical foreign body response to biomaterial, etc.

Intensity of Cell Response

reviewedbyJ.M.Anderson,AnnualReviewMaterialsResearch2001

neutrophils
macrophages
angiogenesis (EC)
monocytes

fused macroph.
fibroblasts
fibrosis

<10 hrs
Acute =
Plasma interactions ...

Time (hours to weeks)

wound
healing
= scar

ba
cte

ria

Phagocyte

RedBloodCells
=SELF

Human cells
Normal vs Leukemic
(CD34+,CD38-,
CD90+,Lin-)

(CD34+,CD38+,
Lin-)

~2-fold

Marrow

Peripheral
Blood

Cord
Blood

anti-CD47

upregulation of CD47 on Ovarian Cancer Cells ~20 yrs ago,

and known then as the OVCAR antigen.

Daily CV1 injections,

200 g.
Daily anti-Her2/neu.

PHAGOCYTOSIS of FOREIGN
Human THP-1 Macrophages eat IgG-opsonized
Sheep-RBC and not Human-RBC

~10x speed

Note the phagocytic forces that deform RBC

TipAdhesionfrequency

Adhesion of human-SIRP depends on Cell Species

Force is similar to
Integrins at
similar rates

SIRP density

70 pN

In the recent SIRP-CD47 co-crystal,

SIRP polymorphisms appear distal to the
CD47-interaction interface.

Hatherley et al Molecular Cell 2008

Polymorphisms postulated to counteract pathogen binding.

All 10 human-SIRP variants displayed & assayed

hum-CD47

hum-SIRP
Variants
displayed as
GFP fusions

. . .

Human-CD47 binding is optimized

for interacting with polymorphic human SIRP

hCD47 binding
SIRP NSG

Self-peptide
v1

30

v2

Estimated 10
Allele Freq.
(Human)
3

Adhesion is
too Strong.

y 1

0.50

11

11 2

, R2=0.85

v9

v6
v4
v8 v3

v10
0.1

0.05 x11

0.3

v7

v5
3

Kd ( M) for hCD47 binding hSIRP Polymorphisms

Signaling is
too weak.

Internalized Beads / Phagocyte

Human-CD47 on polystyrene beads inhibits phagocytosis

THP-1

mCD47
hCD47

Normal HuRBC
(~250 CD47/m2)

1.0

antiCD47block

0.8

Fits:y a b

xm
(Ki x m )
m

0.6

0.4

200

400

600

CD47 Density (molecules/m )

2

800

How does one figure out what proteins

are in a pathway or what proteins are
interacting?

SIGNALING MECHANISM in vitro:

CD47 binds SIRP pSIRP pSHP1
Peptide

dephosphorylates

sustained from 10-45 min

=tail

Myosin
Superfamily

Phospho-Signals
(Rho pathway, SHPs, etc.)

NonMuscle Myosin II

F-actin

Contractile Tension Unit in Stress Fibers

Filament structure based on Sinard, Stafford, Pollard JCB 1989

force
velocity

Efficiency of Phagocytosis proportional to Myosin IIA,

which can be specifically inhibited with a drug in vitro

Self deactivates Phospho-Myosin

and thereby blocks Efficient Engulfment

Tsai & Discher J Cell Biol 2008; Rodriguez et al. Science 2013

How does one figure out what proteins

are in a pathway or what proteins are
interacting?

Mass Spectrometry is the new Nanosensitive, Big Data Tool used for
Protein Discovery & Characterization