What does a protein need to work? Leonid Mirny leonid@mit.edu What - - PowerPoint PPT Presentation

▶
what does a protein need to work
SMART_READER_LITE
LIVE PREVIEW

What does a protein need to work? Leonid Mirny leonid@mit.edu What - - PowerPoint PPT Presentation

Feb 07, 2024 46 likes •398 views

Department of Physics, MIT What does a protein need to work? Leonid Mirny leonid@mit.edu What does a protein need to work? 1. Stable structure 2. Specific active/binding sites. What does a protein need to work? 1. Stable structure 2.

slide-1
SLIDE 1

Leonid Mirny leonid@mit.edu Department of Physics, MIT

What does a protein need to work?

slide-2
SLIDE 2

What does a protein need to work?

  • 1. Stable structure
  • 2. Specific active/binding sites.
slide-3
SLIDE 3

What does a protein need to work?

  • 1. Stable structure
  • 2. Specific active/binding sites.
  • 3. Somewhat unstable structure
  • 4. Non-specific binding site
slide-4
SLIDE 4

A simple DNA-binding protein

Function

  • 1. Find its site on DNA
  • 2. Bind it tightly
  • 3. IF [ligand]>0

leave the site ELSE goto step 1. END

slide-5
SLIDE 5

A simple DNA-binding protein

Function

  • 1. Find its site on DNA
  • 2. Bind it tightly
  • 3. IF [ligand]>0

leave the site ELSE goto step 1. END

slide-6
SLIDE 6

Problem 1: find cognate site among 106-109 non-cognate sites

slide-7
SLIDE 7

Experiment: Riggs et al 1970

kon ≈1010 M−1s−1

Diffusion-limited association Theory: 1D diffusion (“sliding”) + 3D Richter and Eigen 1974, Berg, Winter, von Hippel 1981

kon ≈1010 M−1s−1

WATER CELL ta ~1−10 sec ta ~102 −103 sec

slide-8
SLIDE 8

Model: 1D+3D

..CATGTTCAGGCACGTAGC...

τ1d τ 3d

n

M – genome size

ts– search time

slide-9
SLIDE 9

Model: 1D+3D

..CATGTTCAGGCACGTAGC...

τ1d τ 3d

n

slide-10
SLIDE 10

Our model: 1D+3D

..CATGTTCAGGCACGTAGC...

Energy landscape of 1D sliding

σ

slide-11
SLIDE 11

Protein-DNA interaction energy

DNA-binding domain

DNA

E = e(i,bi)

i=1 l

∑

Energy is strongly sequence dependent NO ENERGY GAP between cognate and random sites Energy landscape

σ

CATGTTTTATATCAGGCACATGCGGCAGTCA

slide-12
SLIDE 12

Roughness of the energy landscape rugged smooth

Results

Fast sliding requires smooth landscape

σ

D

1d ~ e− 7 4(σ /kBT)2

slide-13
SLIDE 13

Roughness of the energy landscape rugged smooth

Results

σ

FRACTION OF TIME SPENT ON THE COGNATE SITE

Specific recognition requires rough landscape

slide-14
SLIDE 14

Either speed or stability but not both !

FRACTION OF TIME SPENT ON THE COGNATE SITE

STABILITY: σ>5 >5kT SPEED σ<2 <2kT

Slutsky.M, Mirny,LA, Biophys J (2004)

Speed-stability paradox

slide-15
SLIDE 15

Proposed Mechanism

slide-16
SLIDE 16

T SEARCH MODE RECOGNITION MODE

Proposed Mechanism

slide-17
SLIDE 17

Experiments

Kalodimos et.al Science.2004

Experiment

slide-18
SLIDE 18

Structure and animation by Babis Kalodimos et al

slide-19
SLIDE 19

Landscape model

ATCATGCATGCCAGTCAGCTCAG Reaction coordinate Reaction coordinate

slide-20
SLIDE 20

Meso-scale dynamics of landscape model

Log(t(x,z))

slide-21
SLIDE 21

Macroscale dynamics

Correlated landscapes Correlated landscapes flat landscape model flat landscape model

103 102 101

Total search time (sec) Folding time (sec) Experimental folding rate

slide-22
SLIDE 22
  • 1. 1D+3D search is fast if the

protein-DNA complex is FLEXIBLE.

  • 2. Conformational transition in the DNA-binding

protein controls the search time.

Summary

slide-23
SLIDE 23

Somewhat unstable structure is needed for …

Function

  • 1. Find its site on DNA
  • 2. Bind it tightly
  • 3. IF [ligand]>0

leave the site ELSE goto step 1. END

 

slide-24
SLIDE 24

What does a protein need to work?

  • 1. Stable structure
  • 2. Specific active/binding sites.
  • 3. Somewhat unstable structure
  • 4. Non-specific binding site
slide-25
SLIDE 25

What does a protein need to work?

  • 1. Stable structure
  • 2. Specific active/binding sites.
  • 3. Somewhat unstable structure
  • 4. Non-specific binding site
slide-26
SLIDE 26

τ1d τ 3d

Model: 1D+3D

..CATGTTCAGGCACGTAGC...

slide-27
SLIDE 27

Model: 1D+3D

..CATGTTCAGGCACGTAGC...

Energy landscape of 1D sliding

σ Εns

slide-28
SLIDE 28

Free energy of non-specific binding more sliding more jumping

Results

σ Εns

Fast sliding requires optimal non-specific binding

slide-29
SLIDE 29

Non-specific binding is needed for …

Function

  • 1. Find its site on DNA
  • 2. Bind it tightly
  • 3. IF [ligand]>0

leave the site ELSE goto step 1. END

slide-30
SLIDE 30

Equilibrium

M~106 - non-specific sites m~10 - number of LacI proteins per cell

P = 1 1+ M m Kd

s

Kd

ns

Kd

ns ≈10−6 M

Kd

s,NO−LIGAND ≈10−12 M

Kd

s,LIGAND ≈10−9 M

Fraction of time the site is bound

P NO−LIGAND = 1 1+ 0.1 = 0.9 P LIGAND = 1 1+100 = 0.01

slide-31
SLIDE 31

Non-specific binding is needed for …

Function

  • 1. Find its site on DNA
  • 2. Bind it tightly
  • 3. IF [ligand]>0

leave the site ELSE goto step 1. END

 

slide-32
SLIDE 32
  • 1. 1D+3D search is fast if the

protein-DNA complex is FLEXIBLE.

  • 2. Conformational transition in the DNA-binding

protein controls the search time.

  • 3. Non-specific binding is essential

for protein function.

Summary

slide-33
SLIDE 33

What does a protein need to work?

  • 1. Stable structure
  • 2. Specific active/binding sites.
  • 3. Somewhat unstable structure
  • 4. Non-specific binding site
slide-34
SLIDE 34
  • 1. Diffusion of a protein on DNA is sequence-

dependent.

  • 2. DNA sequences can influence
  • folded/unfolded equilibrium
  • rate of conf.transition in the protein

(nucleate folding on the target site)

  • 3. Mutations that change the stability and rate

can have affect on the total search time and timing of gene expression.

Testable predictions

slide-35
SLIDE 35

Michael Slutsky, MIT Physics Department of Physics, MIT

Acknowledgements