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Overview of Dynamics. Judith Klein-Seetharaman Co-Course Director jks33@pitt.edu. Objectives of this Lecture. What is dynamics? Time scales of dynamics Methods to study dynamics What data do you get typically? Example: DNA binding. What is dynamics?. What is dynamics?. Definition:

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overview of dynamics

Overview of Dynamics

Judith Klein-SeetharamanCo-Course Director

jks33@pitt.edu

objectives of this lecture
Objectives of this Lecture
  • What is dynamics?
  • Time scales of dynamics
  • Methods to study dynamics
  • What data do you get typically?
  • Example: DNA binding

Molecular Biophysics 3: Lecture 6

what is dynamics
What is dynamics?

Molecular Biophysics 3: Lecture 6

what is dynamics4
What is dynamics?
  • Definition:

= changes in the position of the atoms in a molecule relative to each other or relative to an outside reference point.

http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/relaxdyn

Proteins and other biological molecules are dynamic.

Molecular Biophysics 3: Lecture 6

what are the time scales
What are the time scales?

Molecular Biophysics 3: Lecture 6

example time scales in rhodopsin
Example: Time-scales in rhodopsin

Time-scales here from fs to min

Molecular Biophysics 3: Lecture 6

time scales of protein motions
Time scales of protein motions

Overall tumbling

Libration

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

Time scales from ps to days

Molecular Biophysics 3: Lecture 6

what are the methods to study dynamics
What are the methods to study dynamics?

Molecular Biophysics 3: Lecture 6

nmr parameters and time scales
NMR parameters and time-scales

Overall tumbling

Librations

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

T2, T1r

T1, T2, NOE

HN exchange

J

Chemical shift

Except to some degree in ms-ms range, NMR can report on all time-scales

Molecular Biophysics 3: Lecture 6

other methods and time scales
Other methods and time-scales

Overall tumbling

Librations

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

Fluorescence

IR, Raman

HN exchange with mass spec

Some biophysical measurements are fast…

Molecular Biophysics 3: Lecture 6

trapping of conformations
Trapping of conformations

Overall tumbling

Librations

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

Light

Rapid Mixing

NMR, x-ray

Some biophysical measurements take a long time…

Molecular Biophysics 3: Lecture 6

functions of dynamics and the time scales
Functions of dynamics and the time-scales

Blue: types of motions

Red: functional categorization

Allosteric regulation / global conformational changes

Ligand/protein binding

Chemical kinetics

Catalysis

Overall tumbling

Local folding global

Libration

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

Internal motions are needed to provide flexiblity for functional motions.

Molecular Biophysics 3: Lecture 6

need for protein dynamics in rhodopsin

Trp265

Ala169

Trp265

Ala169

Need for protein dynamics in rhodopsin?

Dark – state structure

Ligand: 11-cis retinal, no clashes

Light-activated structure?

Dark – state structure

Ligand: all-trans retinal, steric clashes

Molecular Biophysics 3: Lecture 6

example function in rhodopsin
Example: Function in Rhodopsin

Definition and Function

Example: Rhodopsin Function in Signal Transduction requires conformational changes

Dark (inactive) Rhodopsin

11-cis retinal

does not bind to G protein

hn

Light-activated Rhodopsin

All-trans retinal

does bind to G protein

involves protein-ligand and protein-protein interactions

Biomolecular motions are needed for function.

Example for function: biomolecular interaction.

Molecular Biophysics 3: Lecture 6

the type of data you can expect
The type of data you can expect

Molecular Biophysics 3: Lecture 6

atomic resolution method example x ray
Atomic resolution method - example X-ray

Gives you snap shots of diffraction patterns in different states

2bcc

1bcc

Zhang, Z.,  Huang, L.,  Shulmeister, V.M.,  Chi, Y.I.,  Kim, K.K.,  Hung, L.W.,  Crofts, A.R.,  Berry, E.A.,  Kim, S.H. Electron transfer by domain movement in cytochrome bc1. Naturev392pp.677-684 , 1998

Molecular Biophysics 3: Lecture 6

more snap shots
More snap shots

1up5 – calmodulin Ca-bound

1ctr – calmodulin free

http://www.molmovdb.org/

http://www.bmb.psu.edu/faculty/tan/lab/gallery/calmodulin.jpg

Molecular Biophysics 3: Lecture 6

any problems
Any problems?

Molecular Biophysics 3: Lecture 6

any problems no information on time scales
Any problems?No information on time-scales.

Molecular Biophysics 3: Lecture 6

time resolved spectroscopy
Time-resolved spectroscopy

Intrinsic Trp fluorescence

Quenching by iodine

Binding of ANS

Dobson,et.al. 1994

Molecular Biophysics 3: Lecture 6

any problems21
Any problems?

Molecular Biophysics 3: Lecture 6

any problems not atomic level information
Any problems?Not atomic level information.

Molecular Biophysics 3: Lecture 6

nmr parameters and time scales23
NMR parameters and time-scales

Overall tumbling

Librations

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

T2, T1r

T1, T2, NOE

HN exchange

J

Chemical shift

Except to some degree in ms-ms range, NMR can report on all time-scales

Molecular Biophysics 3: Lecture 6

chemical exchange
Chemical Exchange

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

Molecular Biophysics 3: Lecture 6

h d exchange can be measured in several ways
H/D exchange can be measured in several ways
  • Slow exchange lifetimes (from mins to days)

by following the loss of HN signal intensity of a protein dissolved in D2O.

  • Faster exchange lifetimes (5–500 ms)

by following the exchange of HN magnetization with that of water protons.

  • At high pH

directly measure the timescale of rate limiting conformational openings

Molecular Biophysics 3: Lecture 6

hd exchange and nmr
HD Exchange and NMR

Hernandez, G., Jenney, F.E.J., Adams, M.W. & LeMaster, D.M.

Proc. Natl. Acad. Sci. USA 97, 3166–3170 (2000).

Molecular Biophysics 3: Lecture 6

relaxation and the noe
Relaxation and the NOE

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

  • Longitudinal relaxation (T1): return of longitudinal (z-component) to its equilibrium value
  • Transverse relaxation (T2): decay of transverse (x,y-component)
  • Heteronuclear NOE

Due to dipole interactions between different nuclei

Molecular Biophysics 3: Lecture 6

from experiments to dynamics data
From experiments to dynamics data

Palmer, A.G., 3rd, M. Rance, and P.E. Wright,.J Amer Chem Soc, 1991

Molecular Biophysics 3: Lecture 6

dynamics in folded unfolded lysozyme
Dynamics in folded/unfolded lysozyme

Unfolded:

Arrows indicate oxidized (all disulfide bonds present) lysozyme

Folded:

Molecular Biophysics 3: Lecture 6

nmr parameters and time scales30
NMR parameters and time-scales

Overall tumbling

Librations

Slow loop reorientation

Fast loop reorientation

Vibration

Side chain rotation/reorient.

S-S flipping

Aromatic ring flips

10-9

10-12

10-6

103

10

10-3

fs

ps

ns

ms

ms

seconds

minutes-hours-days

ms-days: proton exchange

ns-ps: fast internal motions

ms-ms: slow internal motions

T2, T1r

T1, T2, NOE

HN exchange

J

Chemical shift

Except to some degree in ms-ms range, NMR can report on all time-scales

Molecular Biophysics 3: Lecture 6

amplitudes and frequencies
Amplitudes and Frequencies

Molecular Biophysics 3: Lecture 6

popular approach to quantify motions
Popular approach to quantify motions

Measure R1, R2, heteronuclear NOE

“model free” approach

Get order parameter S2 ,τe, τm

Molecular Biophysics 3: Lecture 6

lipari szabo model free approach
Lipari-Szabo Model Free Approach

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

Molecular Biophysics 3: Lecture 6

lipari szabo
Lipari Szabo

http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf

  • Order parameters S2
  • τe, effective correlation function time for internal motions
  • τm, overall tumbling correlation time for global motions

Molecular Biophysics 3: Lecture 6

lipari szabo model free approach35
Lipari–Szabo “model-free” approach
  • Estimate (τm) from R2/R1 for a selected subset of the residues
  • fits to the observed relaxation data using various regression variables
  • model-selection criteria are used to decide which choice is appropriate for each residue
  • Reoptimize using the selected models.
  • Uncertainties in the optimized parameters were obtained by Monte Carlo simulation.

Michael Andrec, Gaetano T. Montelione, RonaldM. Levy Journal of Magnetic Resonance 139, 408–421 (1999)

Molecular Biophysics 3: Lecture 6

example dna binding
Example: DNA binding

Molecular Biophysics 3: Lecture 6

example gcn4 leucine zipper
Example: GCN4 Leucine Zipper

Low S2 indicates high flexibility. S2 can be used to estimate energetics.

Molecular Biophysics 3: Lecture 6

slide38

Energetic Components of Protein-DNA Interactions

Adapted from Jen-Jacobson L., Biopolymers (1997)

The observed free energy (green arrow) for specific binding is the net of large opposing energies.

Molecular Biophysics 3: Lecture 6

slide39

Sources of Entropy and Enthalpy in Protein-DNA Interactions

∆Go=∆Ho–T∆So

Molecular Biophysics 3: Lecture 6

molecular strain

INTER-DEPENDENT

Strain energy

Molecular Strain

When atoms, functional groups or residues (sidechains, bases) adopt positions that are not their own positions of minimum potential energy

Can result from:

Bond bending

Bond rotation

Steric repulsion

Electrostatic repulsion

Strain energy: The energetic cost of strain

Molecular Biophysics 3: Lecture 6

slide41

Thermodynamic parameters

  • H:Enthalpy

measure of heat energy

  • S:Entropy

measure of disorder

  • G: Gibbs Free Energy

G  H – TS

  • C: Heat capacity

measure of the ability of a body to store heat

  • ∆CoP=(∂∆Ho/∂T)P

=T(∂∆So/∂T)P

Using ∆CoP we can calculate ∆Ho, ∆So, and ∆GTat any temperature.

Molecular Biophysics 3: Lecture 6

factors affecting c o p
Factors affecting ∆CoP

free protein + specific DNA ↔ protein-DNA complex

  • ∆CoP is made more positive by:
    • • Burial and desolvation of polar surface
    • • Molecular strain
  • ∆CoP is made more negative by:
    • •Burial and desolvation ofnonpolar surface
    • •Losses of configurational/vibrational freedom
    • (Interface restrains sidechains, bases, backbone)
    • •Restricted freedom of interfacial H2O
    • •Linked equilibria (e.g. protonation, ion binding)

Molecular Biophysics 3: Lecture 6

slide43

Proposal of Spolar & Record(1994)

hydrophobic effect and conformational change

Compared the measured heat capacity changes and the calculated changes in nonpolar ASA and polar ASA

∆CoP is much more negative than predicted.

The “excess” ∆CoP could be accounted for by local folding coupled to binding.

The observed ∆CoP and ∆So could be used to estimate the number of protein residues that fold upon DNA binding.

Equation estimates -1.2 kJ K-1 mol-1 for DSconf

“Conformational changes in the protein that buried large amounts of nonpolar surface are coupled to binding.”

Spolar ad Record, Science (1994)

Molecular Biophysics 3: Lecture 6

example gcn4 leucine zipper44
Example: GCN4 Leucine Zipper

Low S2 indicates high flexibility. S2 can be used to estimate energetics.

Molecular Biophysics 3: Lecture 6

from order parameter to entropy
From order parameter to entropy

Sum of the order paramaters in bound form

… in free form

Result: DS = -0.6 kJ K-1 mol-1

  • Calorimetric data estimates -1.2 kJ K-1 mol-1
  • MD simulations suggest that 40-45% of total conformational entropy loss arises from backbone chain entropy, rest from side-chain
  • NMR result fits well with calorimetric experiment

Molecular Biophysics 3: Lecture 6

slide46

Specific vs. non-specific complexesof the Lac-repressor

Kalodimos et al, Science(2004)

Molecular Biophysics 3: Lecture 6

slide47

Contacts of Lac repressor protein with nonspecific and specific DNA

Kalodimos et al, Chem.Rev.(2004)

“The same set of residues can switch roles from a purely electrostatic interaction

with the DNA backbone in the nonspecific complex to a highly specific

binding mode with the base pairs of the cognate operator sequence.”

Molecular Biophysics 3: Lecture 6

summary of this lecture
Summary of this Lecture
  • Study of biomolecular interactions and dynamics are important to understand function of biomolecules.

Biomolecular interactions

Dynamics

Function

Molecular Biophysics 3: Lecture 6