Protein Dynamics by NMR

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Key Points. • NMR dynamics divided into 2 regimes: fast and slow. • How protein mojons affect NMR parameters depend on whether they are faster or slower.
A vs. B vs. Z DNA, Triplexes and Quadruplexes

Dickerson Dodecamer (bdl001.pdb)

Arnott A RNA Structure (arn0035.pdb)

Comparison of groove width and depth A DNA B DNA

MAJOR MAJOR minor

minor

MAJOR MAJOR minor

Relationship between sugar pucker and helix type B DNA

P O

A DNA

P Base

O

O

Base O

O

7.0 A

P

O

P

5.9 A C3'- endo, 3E

C2'- endo, 2E Orientation of base pairs relative to helix axis 3.4 A

h = 3.4 A

3.4 A

h=3A

Contrasting A and B Helices helical repeat displacement, d helical rise/residue, h tilt twist propellar twist sugar pucker intra chain P-P distance Major groove width depth minor groove width depth

A helix 11 4.4 2.6-3.3 10-20 30-33

B helix 10.5 -0.2 - -1.8 3.4 -6 36-45

C3’-endo, 3E 5.9

C2’-endo and others, 2E 7.0

2.7 13.5

11.7 8.5

11 2.8

5.7 7.5

h

helical repeat

Table: AvsB.doc

Consequences of propeller twisting on local B DNA structure and how DNA responds

minor groove clash at 5'-Py-Pu-3'

major groove clash at 5'-Pu-Py-3'

minimizing minor groove clash at 5'-Py-Pu-3'

Shifting BP over to minimize clashes

Clash due to large twist angle

Clash minimized by decreasing twist angle

Sequence-Dependent Variation of DNA Structure: Callidine’s rules Local DNA structure can be viewed as a result of two opposing effects: 1. The drive to optimize H-bonding and base stacking interactions. 2. The drive to minimize unfavorable VDW interactions between purines that result from the positive propellar twisting of the base pairs at Pu-Py (RY) and Py-Pu (YR) sequences.

DNA compensates for the clashes by both local and coupled responses: 1. Local Responses a. Decrease propellar twist to 0 b. Slide base pair over (reduce δ) 2. Coupled Responses a. Reduce relative twist b. Reduce relative roll angle

Relative magnitude of responses Major Groove

Minor Groove

local responses (at a base pair)

R Y

Y R

propellar twist (always decrease)

-1 -1

-2 -2

δ (open purine δ, decrease pyrimidine δ)

+1 -1

-2 +2

coupled responses (between base pairs)

xRYx

x Y R x

twist angle (always reduce)

+1-2 +1

+2 -4 +2

+1 -2 +1 roll angle (+ value defined as opening in the major groove; changing the central roll angle must be equally compensated by the adjacent roll angles)

-2 +4 -2

εL-εR

+3

-5

240 260 280 300 wavelength (nm)

CD of poly (dG-dC) at pH 7 25 oC Solid line: 0.2 M NaCl Dotted line: after addition of more NaCl

Rich hexamer Z DNA structure (zdf002.pdb)

Rich hexamer Z DNA structure (zdf002.pdb) Minor Groove

Major Groove

Rich hexamer Z DNA structure (zdf002.pdb) CpG step GpC step C3G4-C9G10 G4C5-G8C9

Interstrand base stacking

Intrastrand base stacking

syn anti

Structural Features of Z DNA 1. No major groove, just a surface 13.8 Α wide, 2 A deep. 2. Very deep and narrow minor groove, 3.7 A wide, 8.8 A deep. 3. 18 A wide helix (19 A for B, 23 for A) 4. Interchain phosphate-phosphate distance of 7.7 A; high charge density. 5. 12 bp/turn, 7.4 A/dinucleotide repeat 6. Watson Crick base pairing between C and G. 7. Dinucleotide repeat with an intrastrand GC pi stack at 5’GC-3’ steps and an interstand CC pi stack at 5’-CG-3’ steps. 8. Left-handed stacking at 5’-GC-3’ site with at twist of 50o and -15o twist at 5’-CG-3’ steps. 9. Helix axis is dislocated at 5’-CG-3’ steps. 10. G is in a syn glycosyl conformation, C is in an anti conformation.

Flipping of Base Pairs in B to Z transition TOP VIEW 5'-down O

O

N O

N

5'-up O

N

N H N

anti

O

H H N

O

N H

anti

H

3'-up

O

N

O

Base pair flips by 180o

3'-up

3'-down 3'-down

5'

O

O

O H O

N H

O

N H

N

N

N

N N

O

H N

O O

5'-up H

SIDE VIEW 5' O

O

O

G

glycosyl bond rotates

C

O

O

5'

O O

3' whole nucleoside rotates

O

O

G

C

O O O

3'

Conformational Transition Between B and Z DNA

a a a a a a a a

3'

5' a a a a a a a a

GC CG GC CG GC CG GC CG

5'

B DNA =

3' O

s a s a s a s a

3'

5' GC CG GC CG GC CG GC CG

5'

Z DNA

a= anti glycosyl s = syn glycosyl

a s a s a s a s

3'

A nanomechanical device based on B to Z transtion

Donor and acceptor molecules (fluorescein and Cy3) are attached to a DNA molecule containing a (GC)20 section. When in B form the two dyes are close and show strong FRET, when in Z form, the DNA unwinds by about 3.5 turns, and extends about 6 A, changing the distance by 20-60 A, and greatly lowers the FRET. Nature. 1999, 144-6

Minimum Salt Concentration Required to Form Z DNA Ion (mM)

poly d(G-C)

poly d(G-m5C)

Na+

2500

700

Mg2+

700

0.6

Ca2+

100

0.6

Ba2+

40

0.6

Co(NH3)63+

0.02

0.005

EtOH

60% v/v

20%

Mg2+ + 10% EtOH 4 mM

-

Mg2+ + 20% EtOH 0.4 mM

-

O O P O H

O

H O

O

O

P O

O-

H H N

OH2 O

Mg O H H H2O OH2

H2O

C

O

N N

N

N

H

O

O

Structural factors favoring Z DNA formation CH3 H dR

H

O

N N

N H

N

N H

m 7G

m 5C

electrostatics (Z DNA has higher charge density)

hydrophobicity (methyl group occupies hydrophobic pocket)

O O

N N

N N

O

H

O

RO

dR

O

H

N

H

N

N

CH3

CH3

H N

H N

N

H N

H

RO

bad steric interactions in the anti conformation

N

H

H

N

RO

O

N

N CH3

O

RO

less severe steric interactions in the syn conformation which is the conformation at the purine site in Z DNA

Effect of Substituents on DNA Conformation H

H H dR

O

N N

N H

H N

H

H

N

H

N

N N H

O

dR

N N

N H

O

N H

N

N H

H

2-aminopurine H H dR

O

N N

N H

H N

N

inosine

H

N

H N

O

H N

N H

dR

H

dR

Polymer

C2-NH2 group

Helix

d(A)•d(T)

no

B

d(I)•d(C)

no

B

d(IIT)•d(ACC)

no

B

d(AG)•d(CT)

yes

B, A

d(AGC)•d(GCT)

yes

B, A

d(GC)

yes

B, A, Z

d(GT)

yes

B, A, Z

d(2AP-T)

yes

B, A, Z

O

dR

Metal cations bind in the minor groove with water Structure of the potassium form of CGCGAATTCGCG: DNA deformation by electrostatic collapse around inorganic cations. Biochemistry. 1998 Dec 1;37(48):16877-87.

Figure 1 The fused hexagon motif of A-tract DNA. The four layers are coded by color with the primary layer light blue, the secondary layer magenta, the tertiary layer blue, and the quaternary layer red. The fused hexagon motif is shown in space filling representation, with van der Waal radii of oxygen atoms. (a) Stereoview into the minor groove of the DNA. The DNA is colored by CPK and shown in stick representation. (b) View across the groove, approximately down the normal of the central hexagons. Sites of potassium occupancy are indicated by plus signs. The DNA bases are shaded. Base functional groups that interact with the fused hexagon motif are indicated by circles. (c) The geometry of the sodium form fused hexagon motif. Distances are in red and angles are in white.

X-ray crystal structure of a 1:1 complex of netropsin: DNA

Loss of O2 carbonyl disrupts spine of hydration

The role of minor groove functional groups in DNA hydration. Nucleic Acids Res. 2003 Mar 1;31(5):1536-40.

Conformational and Electrostatic Factors Favoring Various Forms of DNA or RNA B Form of RNA

P O

A Form of RNA

P O

Base O

Base

bad steric interaction

O

O

P

P OH

C3'- endo, 3E

C2'- endo, 2E

O P

H

O

Z DNA intrastrand phosphate hydration

H

O

O

Z: 5.6 A

P

H

O H

O

O

A: 5.7 A B: 6.7 A

steric interaction less severe

O

OH

Z: 6.2 A

P

O

Evidence for Triplex Helix Formation From Mixing Experiments Monitored by UV

nd(T)10d(A)10

d(T)10 + d(A)10 A260 1

A260 1

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0

50 molar % dT10

100

0

50 66

100

molar % dT10

Analysis of mixing curves of nucleic acids by UV relies on the hypochromic effect observed upon formation of stacked base pairs

Polypyrimidine Triplex Motif H CH3

N

O

N

O

parallel helix, Hoogsteen base pair

H H

H

Major groove O

N H

N N

CH3

H N

N

H N

N O

H

antiparallel A helix Watson Crick base pair 5' - TTTTTTTTTT- 3' 5' - AAAAAAAAAA- 3' 3' - TTTTTTTTTT- 5'

WatsonCrick

Hoogsteen

H H

N

N

N H

O H

H

H O

N N

N H N N H H

WatsonCrick

protonated C required for base pairing H H N

H

N

H N

O

5' - CCCCCCCCCC- 3' ++++++++++ 5' - GGGGGGGGGG- 3' 3' - CCCCCCCCCC- 5'

Hoogsteen (+ indicates H +)

Polypurine Triplex Motif N N

N

anti parallel helix, Reverse Hoogsteen base pair

H N

N

H H

Major groove H N

N N

O

H H

N

CH3

N

H N

N O

H

antiparallel A helix Watson Crick base pair 3'-AAAAAAAAAA-5' 5'-AAAAAAAAAA-3' 3'-TTTTTTTTTT-5'

WatsonCrick

Reverse Hoogsteen

N N

N

O

H

H H

H

N

N

O

N N

N H N N H H

WatsonCrick

H H

N

H

N

H N

O

3'-GGGGGGGGGG-5' 5'-GGGGGGGGGG-3' 3'-CCCCCCCCCC-5'

Reverse Hoogsteen

Intramolecular Triplex NMR structure 1gn7.pdb

Intramolecular Triplex (1GN7.pdb) highlights

Yeast phenylalanine tRNA (4tna.pdb)

acceptor end triplex region

anticodon

Yeast phenylalanine tRNA (4tna.pdb)

acceptor end

anticodon

Base Pairing Found in tRNA Η

Ν CH3

Η Ν

Ο

Ν

CH3

Η

Ν

Ν

Ν

Ο Ο

Ν

Ν

Ν Η

Ν

Ν

Ο

T54

Η

m 1A58

Η Ν Ν

Ο

Ν Η

U69

G4 Ο

Ο Ν

Η

Ν Ν

Ν

Ν

Ν

Ν Η

Η

G18

Ν

Ο H

Ν

N

CH3 Ν

Ν

A9

Ν

Η Ν

Ν

Ν

Ν Ν

Ν

Ο

Ν

Ο Η Ν

Ν Η

U12

Η Η Ν Ν Ν

Ν Ν Η

Ο

A23

Η

Η

Η

Η

Η Ν Η

Ν

m 7G46 Ν

Ν

Ο

Η Ν

Η

C48 Ν

Ν

Η

Ν Ν

Η

O

Ν Ν

Ν

G15

H

ψ55

Ν

Ο

Η

Η

G22

Ο

C13

Chemical Probe Assays of Nucleic Acid Structure and Interactions Hyper reactive sites

Accessible sites *P

*P

*P

*P

*P

induce cleavage

induce cleavage *P

*P

*P

A

B

C

D

A. B. C. D.

- binder + binder pre conformational change post conformational change

Mechanism of strand cleavage following glycosidic bond hydrolysis

Base

B

hydrolysis

O O O

O P

-

O

O P

enolization

O

H O

H

O P

O

O

O

O P

-

O P

O

O

O

β-elimination (retro-Michael Rxn)

O P

H H O O

-O

H

O

B

O

H O

O

P

O

O

B

O P

O -

O

O

O H H O

O

O -

-

H

β-elimination (retro-Michael Rxn)

O

O

H

H

-

-

O

O-

O

O

hemiacetal abasic site

O

-

O

O

B

H

O

O P

O -

O

H

O

O

O P

O

-

O

O P -

-O

O P

O

O

O

3'-phosphate

5'-phosphate

In the presence of piperidine, the β-elimination reactions may take place through the enamine.

-

Dimethyl Sulfate Probe for the Accessibility of N7. A Major Groove Accessibility Probe. CH 3

O O S O CH 3 O

CH 3

N

N

N

DMS

N

G>A

Dimethyl sulfate (DMS). Alkylates sterically accessible N7 of purines. (See the Maxam-Gilbert G reaction in chapter on sequencing.) Reactivity: 1. The N7 of G in single strand and duplex DNA. 2. The N7 of purines in the anti conformation.

Diethylpyrodicarbonate (DEPC) probe of single stranded purines O O CH3

O

O

O O

O

CH3 +

N

N

N

N

DEPC A or G Approach is from the major groove side. DEPC is bulky, and reaction with N7 is inhibited in duplex DNA. Reactive N7: 1. Single strand DNA. 2. Loops of cruciforms. 3. Purines in the syn conformation in Z DNA.

Osmium tetroxide and KMnO4 oxidation of 5,6-double bond of pyrimidines O H O

CH3 O N O

N

H

Os N

H

O O N

N

O

O CH 3 O N O Os N

H

O

O

N

Reagent must approach from above or below the plane of the pyrimidine therefore it will not work well on stacked DNA Reactivity: 1. Reacts with T's at junctions between B & Z DNA. 2. Reacts with T's in cruciform loops.

Hydroxylamine reactions with C HO

NH2 N N

HO

N N

O

N

N

H O

N HO N

N

H O

H

labile to piperidine

Reagent must approach from above or below the plane of the pyrimidine therefore it will not work well on stacked DNA Reactivity: 1. Reacts with C's at junctions betwee B & Z DNA. 2. Reacts with C's at junctions between out of phase Z DNA blocks such as the sequence shown below: 5'-GCGCGC-CGCGCG-3' 3'-CGCGCG-GCGCGC-5' 3. Reacts with C's in cruciform loops.

H DNA Structure 5'

3'

single strand GAAGGA

triplex

5'3'-

5'3'-

CTTCCT GAAGGA CTTCCT

AGGAAG TCCTTC

GAAGGA CTTCCT

TCCTTC AGGAAG TCCTTC

triplex

AGGAAG

5' 3'

single strand

5' 3'

3' 5'

Chemical Probing of H-DNA Johnston, B.H. (1988) The S1-sensitive form of d(CT)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science, 241, 1800-4.

a) normal superhelical density b) Higher superhelical density (higher torsional stress)

The End Replication Problem

Succesive rounds of replication lead to progressive shortening of the ends of DNA

Telomerase solves the End Replication Problem

ribonucleoprotein

elongation

translocation

Schematic structure of a telomere

Annu. Rev. Pharmacol. Toxicol. 2003. 43:359–79

The G’s in the telomere sequence can form Quartets via Hoogsteen Base Pairing,

• Hoogsteen base pairing leads to parallel strands if glycosyl bonds are anti • Center of quartet has large negative electrostatic potential H

H N

N

N

N

H

N H

N H

H N

N H N

N

N

+ O

N

N

O

H

H

N H

O

N

H N

H

H

N H H

N

O N

N H

Four possible orientations of Gn strands

parallel

antiparallel

mixed (3+1)

antiparallel

Ways of forming intramolecular quadruplex formation with [GxNy]z with 3 types of loops: propeller, lateral, diagonal propeller

hybrid

3'

lateral loop

3'

external or propeller loop

external loop

5' 5' lateral loop

chair

lateral loop

basket

lateral loop

lateral loop

lateral loop

3' 3' 5'

5' lateral loop

diagonal loop

Glycosyl conformation depends on strand orientation. Bases in one base quad can all flip from anti to syn, and syn to anti a

a

a

a

s

a

a

a

a

a

s

a a

a

s

s

a

s

s

a

a

a

a

a

Flip central Base quad s

a a

s

a

s

s

a

a

s

s

a

a a

s a

s

s

a

s

a

s

a

s

Front. Chem. 4:38. doi: 10.3389/fchem.2016.00038

J. Phys. Chem. B, Vol. 110, No. 32, 2006 16077

Characterized human telomeric DNA Gquadruplex structures lateral 3

1

T20

T8

A3 A21 G4

syn A9

G10

T8

G11

G17

G6 T7

G12

G16

G23

G12

T20

G22

G4 T13

A15

T13

T14

2

A3

2

lateral

hybrid-1 lateral

T8

T18

T7

Na

G10

T8 T20

T19

A9

G6

G10

syn

G6 G11

Na+ G17

T7 A21

+

G18

diagonal

lateral

A9

A21

syn

T14

hybrid-2 lateral

T19

G11

G5

A21 A15

G6

T19

G5

1

G10

syn

anti

G24

propellar

anti

G18

T7

3

T19

A9

anti

G18

K

+

G17

G5

G5

G11

G4 G12

G12

Na+ G16

G16

G4 T13

A15

X Y A15

5'

3' T14

T14

diagonal

basket

diagonal

form 3

T13

Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex

J. Mol. Biol. (2014) 426, 1629–1650

Chemical Probes of G-quartet structures. Hoogsteen base pairing interferes with G reaction (reaction at N7).

PNAS 2002 99 11593-11598