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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

Triple resonance experiments

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

Restrained Molecular Dynamics Simulation

Distance Geometry

IUPAB NMR Workshop 2009

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 ?

IUPAB NMR Workshop 2009

Challenges

Dynamics in proteins

Unfolded and partially folded proteins

Protein folding

Large protein assemblies

IUPAB NMR Workshop 2009

Energy LandscapeEnergy

Denaturant stabilized

IUPAB NMR Workshop 2009

Folded HIV-Pr Unfolded HIV-Pr

IUPAB NMR Workshop 2009

F3 (HN)

F1(N)

F2(N)

3D spectrum with two 15N axes

H

N

2D3D

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

F3

F1

F2

NH

15N

15N

HNN

IUPAB NMR Workshop 2009

F3

F1

F2

i

i -1

i +1

i

NH

F1

15N

IUPAB NMR Workshop 2009

F3

F1

F2

i

i -1

i +1

i

i

NH

F2F1

15N 15N

NH

Triplet filter through HSQC

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

F2 (N)

F1(N)

F3 (HN)

i-1

ii-1 i

i+1

i

F2 (N)

F1(N)

F3 (HN)i

HN(C)N

IUPAB NMR Workshop 2009

IV

HN

NH

N(C

)N

Special Peak patterns in HNN and HN(C)N

IUPAB NMR Workshop 2009

HNN planes

IUPAB NMR Workshop 2009

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)

IUPAB NMR Workshop 2009

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)

IUPAB NMR Workshop 2009

Tuning the HNN

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

Unfolded HIV-I Protease

IUPAB NMR Workshop 2009

Structural propensities

• Secondary Chemical shifts:

δs = δobs – δrc

Cα Hα CO δs + - + α− helix

- + - β- sheet

IUPAB NMR Workshop 2009

Secondary chemical shifts

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

6M Gdn 5M Gdn

IUPAB NMR Workshop 2009

IUPAB NMR Workshop 2009

Folding propensities in HIV-1 protease

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

IUPAB NMR Workshop 2009

SUMO

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

IUPAB NMR Workshop 2009

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

Line broadening : micro-milli second time scale conformational transitions

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

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

NMR Insights into Protein Function

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

Protein Dynamics

Tumbling motions

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

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

Functional significance

IUPAB NMR Workshop 2009

Active site

HIV-1 protease

Structure – Dynamics - Function

IUPAB NMR Workshop 2009

Heavy Heavy ChainsChains

Intermediate Intermediate ChainsChains

DLC8DLC8

Dynein Light Chain Protein (DLC8)

DLC8DLC8 – – DimerDimerpH 7pH 7

DyneinDynein

MicrotubuleMicrotubule

CargoCargoMoleculeMolecule

IUPAB NMR Workshop 2009

Dynamic regions

Dynamics: R2, Curved temp dependence, Line broadening

IUPAB NMR Workshop 2009

Dynamics, pH 7

Dynamics, pH 6

IUPAB NMR Workshop 2009

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

IUPAB NMR Workshop 2009

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