Transcript Protein insertion into membranes: production, purification, and

Welcome to
2011 UK CD and LD winter
workshop
Thanks to MOAC, Department of
Chemistry, and the University of
Warwick for hosting us.
Safety
In the lab: lab coats, safety spectacles,
gloves as appropriate.
In the event of MOAC fire alarm go
outside the front door.
In event of chemistry fire alarm – we
meet outside library. No lifts!
Elsewhere read signs.
Where will we be??
Lectures and computer workshops:
MOAC EPSRC Doctoral Training Centre
Laboratory work:
Mainly chemistry
Food:
Lunches: DIY – including clear-up! Use the time to
get to know other people.
Coffee/Tea/Squash: available – again DIY.
Dishwasher need stacking with dirty dishes and
emptying when clean!
Tuesday dinner with, posters etc. in MOAC.
Wednesday: Browns in centre of the city
More things
Keep the common room tidy
READ but not REMOVE books!
Internet access via the common room
computers. Ask about wireless if you
have a laptop.
Wear your name badges.
Receipts/refunds on deposit see me of
you haven’t got one.
People
All organisational credit to:
Anne Maynard and Fiona Friel
Big problems: Alison Rodger
Warwick students
Lecturers and course leaders – see programme.
Circular Dichroism
Spectroscopy
Introduction
Alison Rodger, University of Warwick
[email protected]
Absorbance
Bonds  Molecules  Spectroscopy
Molecules are 'glued together' by electrons between
the atoms. ~ Two electrons per bond.
Two types of bonds are important for bio molecules:
s (sigma) bonds look like s-orbitals
when viewed along the bond axis.
 (pi) bonds look like p-orbitals (dumbbells)
when viewed down the bond axis
Also non-bonding pairs, e.g. lone pairs on N & O
Ultra violet/visible (UV/vis): how much energy is
required to push electrons to new orbitals.
Electrons  bonds  structure
UV/visible light: l ~ 180 nm – 800 nm, energy hn = hc/l
causes electrons to go E
UV: –350 nm
Vis: 400 nm –
to higher energy levels.
Excited electronic state
l required depends on electron
rearrangement
needed.
.
In solution: broad bands due to
diff. vibrational levels
in excited state &
Ground electronic state
molecules having slightly
different energy levels.
r
Ground vibn’l level
Absorption spectra of DNA &
proteins
With proteins and DNA the transitions we usually
study
are n  * and   * so can access them with
normal spectrometers.
Below 200 nm need nitrogen purging because O2
absorbs
Absorbance: A = log(Io/I)
= log(intensities in/out)
Beer Lambert A = ecl, e extinction
coefficient (units?),
c concentration, l length (cm)
Linearly polarized light
• All photons in a beam of light have an electric field vector,
E, that is at right angles to the direction of travel of the
x
beam, x, and varies as a sine wave
E
Before polarization:
unpolarized beam
E
E
polarizer
E
After polarization:
linear polarized beam
Circularly polarized light
• Add two linearly polarized
light beams - both
propagating along x
• y-polarized +
• z-polarised but starting ¼
wavelength out of phase
from the y one.
Ey= msin(2y/l)
Ez= mcos(2z/l)
The two waves add together to form right handed (clockwise)
circularly polarized light.
y
y
z
z
x
or time
Circular Dichroism
• CD is the difference between the absorption of left
and right handed circularly polarized light as a
function of wavelength.
A
CD
• The difference very small (~<<1/1000 of total)
 DA(l) = AL(l)-AR(l) = [eL (l) - eR (l)]lc or
 DA(l) = De (l)lc
• De ~ typically < 10 M-1cm-1 vs. e ~20,000 M-1cm-1
CD is very small difference between two large signals
Circularly polarized light
Linearly polarized light:
Electric vector direction constant—magnitude varies.
Circular polarized light:
Electric vector direction varies—magnitude constant
CD Spectra
Varying absorption of circularly polarised light by chiral
molecules results in distinctive spectra under
absorption bands
Need chiral light and chiral molecule to get CD spectrum
of a solution
CD spectropolarimeter
Xenon
lamp
M = mirror
P=prism
Max light intensity: 300-400 nm
Monochromater
prism
Quartz 1/4 lplate. Oscillates
at ~ 50 Hz
 CPL
CD=DA/(absorbance units)=4(degrees)/(180ln10)
CD=(millidegrees)/32,980
Empirical analysis of CD
HPLC detector
Structural change
CD to answer: does it change
structure?
Circular dichroism spectra of the plant defensin Hs-AFP1 in
water/acetonitrile (1:1, v/v) + 1% formic acid
10
Unreduced Plant defensin
5
Chemically reduced plant defensin
(m d e g )
0
190
195
200
205
210
215
220
225
230
235
-5
-10
-15
-20
Wavelength (nm)
Reduced and unreduced plant defensins
240
The CD signal for a protein depends on
its secondary structure
Two regions
Secondary structure (170 – 260 nm)
Aromatic region (240 – 360 nm)
Secondary structure
- Amide backbone
a-helix
20
15
b-sheet
10
turn
5
0
-5
10
180
Poly
proline
200 type II 220
Wavelength/nm
240
Aromatic
- Phenylalanine (250 – 270 nm)
-Tryptophan (260 – 300 nm)
- Tyrosine (270 – 290 nm)
- Disulfide bonds
a-helical protein spectra are
distinctive:
222,208,~190 nm
260
Amino acids,
peptides
and proteins
H
H
CD  secondary structure of
protein
• Fit the unknown CD curve u to a
combination of standard curves
• In the simplest case use the
standard spectra for secondary
structures
t = xaa + xbb + xcc
• Vary xa, xb and xc to give the
best fit of t to u
•
while xa+ xb + xc = 1.0
CD spectra can be analysed by the
structure-fitting program “cdsstr” • fits best with:
(of C.J. Johnson) to obtain % of
xa= 80%; xb=0%; xc= 20%
secondary structure motifs.
• agrees well with
Cdsstr uses a basis set of protein
structure:
spectra
78% helix, 22% other
CD signals are sensitive to secondary
structure: coiled-coil (2 a-helices twisted)
CD signals for GCN
O'Shea et al. Science (1989) 2
figure 3: 34M GCN4-zip in 0.15
10mM phosphate pH 7.0
80
70
g cm2dmol-1
60
50
40 repeat
Characteristic heptad
abcdefgabcdefgabcdefgabcdefg
30
MKQLEDKVEELLSKNYHLENEVARLKKL
Salt bridge
O
hydrophobic 0 C
50 OC
75 OC
The CD signal for a protein depends on
its secondary structure
—— chymotrypsin (~0.1 helix, 0.15 b-sheet,
0.15 b-turn)
—— lysozyme (mixed 0.4 helix, 0.2 turn)
—— triosephosphate isomerase
(mostly a some b)
—— myoglobin (all a)
Protein conformation as a function of
environment
Transmembrane
SPP Hydrophobic
TPP
N
a-helix
region
5
3
Molar Residue ellipticity
Pre-PsbW, thylakoid
membrane protein
Tris buffer soluble Pre-PSbW
and and Unfolded using
Guanidinium Chloride
4
2
1
0
190
200
210
220
230
-1
-2
-3
Wavelength / nm
‘No structure’ in
guanidinium chloride
Some in tris buffer
(multimer)
Lots in SDS micelles
(folded, 2 helices)
240
250
260
C
CDsstr results for pre-PsbW
60
50
40
Helical
Number of
30
residues
- Strand
b -Turn
Other
-
20
10
0
Pre-PSbW
(SDS)
Pre-PSbW (OG)
PSbW (OG)
Lots of a-helix
Averaged CD spectrum of the sample 67544
40
30
CD/mdeg
20
10
0
190
-10
210
230
250
270
290
310
1.4 mg/mL; 0.01 cm;
42% α-helix
208 nm, 100% a-helix = 12
mol-1dm3cm-1
-20
-30
Wavelenghth/nm
Antibody
0% α-helix
10% other helix
33% β-sheet
14% turn
42% other
Largely b-sheet protein
dE spectra of samles 71603-71638 using a 0.1mm cuvette
dE (mol-1 dm 3 cm -1)
2
1
0
-1
-2
-3
-4
258
254
250
246
242
238
234
230
226
222
218
214
210
206
202
198
194
190
-5
Wavelength (nm)
0.01 cm;
0.3 mg/mL;
16% PPII
19% β-sheet
15% turns
47% other
Typical mixed a-b-sheet protein spectrum
Wavelength (nm)
258
254
250
246
242
238
234
230
226
222
218
214
210
206
202
198
194
5
4
3
2
1
0
-1
-2
-3
190
dE mol-1 dm 3 cm -1
dE - Sample 71004 subtracting the pH 7.2
buffer baseline
15% α-helix
11% other helix
26% β-sheet
12% turns
36% other
Near UV: protein CD
CD requires helical electron
R = Im  0 μ f    f
motion
m 0
Require magnetic dipole transition moment  0
Require electric dipole transition moment  0
CD from coupled oscillators
R = CD strength
= Im(.m)
=electric dipole
transition moment
m=magnetic dipole
transition moment
-* transition
of a helical polypeptide
Low energy, 208 nm
High energy, 190 nm
Oriented CD spectra
Vancomycin & ristocetin
Glycopeptide antibiotics that prevent cross-linking and
transglycosylation during bacterial cell wall formation.
Noncovalent dimerisation plays a key role in their activity
CD used to give binding constants V-V, V-R and
V-peptides, R-peptides. Assume non-covalent dimers.
Circular dichroism spectra for the Titration of Vancomycin with Ristocetin
CD change (induced CD)
a [dimer]
50
40
V + R V-R
30
20
In te n s ity (m d e g )
10
0
190
210
230
250
270
-10
[25uM vanco + 25uM risto]
-20
[50uM vanco + 25uM risto]
[75uM vanco + 25uM risto]
-30
[100uM vanco + 25uM risto]
[125uM vanco + 25uM risto]
-40
-50
[150uM vanco + 25uM risto]
[175uM vanco + 25uM risto]
[200uM vanco + 25uM risto]
-60
Wavelength (nm)
290
310
Kdimerisation= 205 (mM)-1
Nucleic acid CD
DNA and RNA polymers: sugar units of the backbone
provide the chirality, but not the chromophores.
CD spectrum of a polynucleotide arises from interaction
between the -* transitions of
stacked bases.
But note isolated nucleotides
are chiral
Use CD to identify which
polymorph: CD varies more with
base orientation than sequence
Nucleic acid CD spectra
B-DNA:
72%, 50%
& 31% GC
content
Calf thymus DNA:
B-DNA (10.4 bases)
A-DNA
B-DNA (10.2 bases)
Poly[d(G-C)] 2:
B-DNA
A-DNA
Z-DNA
B-DNA: 275>0, 258=0, 240<0, 220>/=0, 180/190>>>0
A:DNA: 295</=0, 260>>0, 250-230>/=0, 210<<0, 190>>0
Z-DNA: 290<0, 260>0, 195/200<<<0, 185 -180=0
RNA: CNG repeats (neurological
disorders e.g. Myotonic Dystrophy )
Which is
melted?
Unusual RNAs: adopt duplex A-form plus something else.
??? Triplex.
3'
5'
[Ru(phen)3]2+
ct+D
high r
ct+D
low r
AT
GC
