iupab nmr workshop 2009 exploration of the protein world: …iupab/ws_nov26_2009_4.pdf · 2010. 1....

IUPAB NMR Workshop 2009

Exploration of the protein world: Challenges and NMR solutions

R. V. Hosur

Department of Chemical SciencesTata Institute of Fundamental Research

Mumbai 400 005

MBU, June 20, 2008


C. M. Dobson, Nature 426, 884-890 (2003)

The Protein World: A Structural Biology perspective

The proteome contains a

large number of intrinsically

unfolded proteins


Triple resonance experiments


F2 (Cα)

F3 (HN)

i+1i-1 i

F2 (Cα)

F1 (N)

HNCA

F3 (HN)F3 (HN) F3 (HN)

ii

i+1

i-1i-1

i-2


F2 (Cα)

F3 (HN)

i+1i-1 i

F2 (Cα)

F1 (N)

F3 (HN)F3 (HN) F3 (HN)

HN(CO)CA

i-2

i-1i


Structure Calculation • Distance Restraints

• Energy Function E = Ef + ENOE

i i il uj j jr r r< <

2 2 - + - ( ) ( )l ul u

NOE ij ij mn mnij mnij mn

k kE r r r r= ∑ ∑

= 0; if the restraint is satisfied


Restrained Molecular Dynamics Simulation

Distance Geometry


Protein FoldingLandscape Perspective

Idealized funnel landscape Rugged energy landscape Unfolded ensemble

Intermediate ensemble

Native ensemble

How to monitor the continuous conformational changes as the protein folds ?


Challenges

Dynamics in proteins

Unfolded and partially folded proteins

Protein folding

Large protein assemblies


Energy LandscapeEnergy

Denaturant stabilized


Folded HIV-Pr Unfolded HIV-Pr


F3 (HN)

F1(N)

F2(N)

3D spectrum with two 15N axes

H

N

2D3D


An Efficient High-Throughput Resonance Assignment Procedure for StructuralGenomics and Protein Folding Research by NMR

Neel S. Bhavesh, Sanjay C. Panchal, and R. V. Hosur*Biochemistry 40, 14727-14735, 2001.

Accelerated Publications

Improved 3D triple resonance experiments, HNN and HN(C)N, for HNand 15N sequential correlations in (13C, 15N) labeled proteins: Application

to unfolded proteinsSanjay C. Panchal, Neel S. Bhavesh & Ramakrishna V. Hosur∗

Journal of Biomolecular NMR, 20: 135–147, 2001.

A novel protocol based on HN(C)N for rapid resonance assignmentin (15N, 13C) labeled proteins: implications to structural genomicsq

Amarnath Chatterjee, Neel S. Bhavesh, Sanjay C. Panchal,and Ramakrishna V. Hosur,*

Biochemical and Biophysical Research Communications 293 (2002) 427–432


C N C C N C C N C

O

O O

C

C

CH

H H

α

α

α

β

β

β

i-1 i i+1

HNN

t1

t3t3

t2t2

t2

t3


F3

F1

F2

NH

15N

15N

HNN


F3

F1

F2

i

i -1

i +1

i

NH

F1

15N


F3

F1

F2

i

i -1

i +1

i

i

NH

F2F1

15N 15N

NH

Triplet filter through HSQC


C N C C N C C N C

O

O O

C

C

CH

H H

αα

α

β

β

β

i-1 i i+1

HN(C)N

t1t2t2

t3

t3


F2 (N)

F1(N)

F3 (HN)

i-1

ii-1 i

i+1

i

F2 (N)

F1(N)

F3 (HN)i

HN(C)N


IV

HN

NH

N(C

)N

Special Peak patterns in HNN and HN(C)N


HNN planes


Sequential walk through HNN spectrum.

(G) (X) (Z)F 1 (

15N

)

F3 (HN)

G

X

G

X

Z

X

Z

B

-PGXZB-

Neel S. Bhavesh, Sanjay C. Panchal and Ramakrishna V. Hosur. Biochemistry 40, 14727-14735, (2001). (Accelerated publication)


Alanine Check Points in HNN and HN(C)N SpectraAmarnath Chatterjee, Ashutosh Kumar and Ramakrishna V. Hosur*

J. Magn. Reson. 181, 21-28 (2006)

Tuning the HNN Experiment: Generation of Serine-Threonine Check pointsJeetender Chugh, Dinesh Kumar and Ramakrishna V. Hosur*

J. Biomol. NMR, 40, 145-152 (2008)


Tuning the HNN


G63 G63

A11 A11A21 A21

F62 F62

F64 F64

N10 N10

D12 D12

V22 V22

D20 D20

Chatterjee et al, J. Magn. Reson. 181, 21-28 (2006)

Alanine residues behave like glycines in HNN


Chugh et al J. Biomol. NMR, 40, 145-152 (2008)


1. HIV-1 protease : 22 kD1. HIV-1 protease, unfolded : 22 kD2. HIV-1 PR precursor : 18 kD (intrinsically unfolded)3. HIV-1 PRTD precursor : 29 kD 4. FK 506 binding protein (FKBP) : 12 kD1. Small ubiquitin like modifier (SUMO): 10 kD1. Small ubiquitin like modifier (SUMO), unfolded : 10 kD2. Drosophila SUMO (folded, unfolded)3. Barstar, unfolded : 10 kD4. Barstar, aggregated (molten globule at low pH) : 160 kD1. GTPase Effector Domain (GED):15 kD, oligomerises (>5

Mda)1. DLC8: dyenin light chain, 10 kD folded2. DLC8 unfolded

Protein systems investigated


HIV-I Protease

• 22 kDa Homodimer

• 99 amino acids each

• Helps in maturation of progeny viruses

• Target for anti AIDS drug using protease inhibitor

Flaps

PDB id 1GSL

Active Site

Dimerization domain

Hinge


Unfolded HIV-I Protease


Structural propensities

• Secondary Chemical shifts:

δs = δobs – δrc

Cα Hα CO δs + - + α− helix

- + - β- sheet


Secondary chemical shifts

Neel S. Bhavesh, Sanjay C. Panchal, Rohit Mittal and Ramakrisna V. Hosur. FEBS Lett. 509, 218-24, 2001.


Residual structural propensities derived from the NMR data are displayed on the crystal structure of HIV-1 protease tethered dimer.

β-structure

α-helixNeel S. Bhavesh, Sanjay C. Panchal, Rohit Mittal and Ramakrisna V. Hosur. FEBS Lett. 509, 218-24, 2001.

Local structural preferences: Initial folding events


6M Gdn 5M Gdn


Folding propensities in HIV-1 protease

∆R2 = 3.0 – 4.5 s-1 ∆R2 = 1.5 – 3.0 s-1


SUMO

Mishra, et al, J. Biol. Chem. 279, 31445-31454 (2004)


Ashutosh Kumar et al, Biophys. J. 90, 2498-2509 (2006)Ashutosh Kumar et al, J. Mol. Biol. 361, 180-194 (2006)

Equilibrium populations down the funnel

Secondary shifts

8M urea

7M urea

6M urea

5M urea

4M urea

3M urea


Line broadening : micro-milli second time scale conformational transitions

Ashutosh Kumar et al, J. Mol. Biol. 361, 180-194 (2006)


N

N

C

1M0M

8MN

C

N

C

7M 6MN

C

5M

C

N

4M

C

3M

C

N

C

N

Ashutosh Kumar et al, J. Mol. Biol. 361, 180-194 (2006)

Equilibrium populations down the funnel


NMR Insights into Protein Function


Random Coil

Pre molten Globule

Molten Globule

Folded

Structure Function Paradigm

Random Coil

Pre-Molten Globule

Molten Globule

Folded

AggregatedUversky V. N. (2002) Prot. Sci. 11, 739


Protein Dynamics

Tumbling motions

Internal high frequency motions (nano- to pico- second time scales)

Conformational transitions (milli- to micro- second time scales)

Functional significance


Active site

HIV-1 protease

Structure – Dynamics - Function


Heavy Heavy ChainsChains

Intermediate Intermediate ChainsChains

DLC8DLC8

Dynein Light Chain Protein (DLC8)

DLC8DLC8 – – DimerDimerpH 7pH 7

DyneinDynein

MicrotubuleMicrotubule

CargoCargoMoleculeMolecule


Dynamic regions

Dynamics: R2, Curved temp dependence, Line broadening


Dynamics, pH 7

Dynamics, pH 6

IUPAB NMR Workshop 2009DLC8 binding with (VYTKQTQTTST)

Free protein, pH 7

Spectrum at pH 7,DLC8-peptide complex

Spectrum at pH 6,Free protein + complex

Recognition motif(K/R)XTQT

pH Switch for cargo trafficking


Ph. D. Students

Sanjay C. PanchalNeel S. BhaveshAmarnath ChatterjeeAshutosh KumarJeetendra ChughP. M. KrishnamohanManoj RoutDinesh KumarSwagata Chakraborty

Post-docs

Ragini SinhaManeesha BarveShilpi Sharma

Others

Dr. Rohit MittalJyoti Ranjan MishraRam Kumar MishraAnindya Ghosh Roy

Dr. M.V. HosurDr. B. Pillai

iupab nmr workshop 2009 exploration of the protein world: …iupab/ws_nov26_2009_4.pdf · 2010. 1....

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