Download Ser Trp Thr His Glu Asn Gly Lys His Val Trp Arg

Andrew Rylaarsdam and Douglas A. Vander Griend, Ph.D., Department of Chemistry & Biochemistry, Calvin College
Folding Stability
Biomolecular folding is a type of molecular self-assembly in
which self-interaction drives a molecule to its native
conformation. We synthesized six 12-amino acid β-hairpin
peptides stabilized by π-π interactions between the indole
groups of two tryptophan residues. Histidine binding pockets
of varying stability are incorporated closer to the β-turn.
Folding stability and binding affinity were tracked via NMR,
MALDI and UV-Vis spectroscopy and the data modeled using
factor analysis methods. Comparative thermodynamic details
of the binding events are presented, including binding
constants and spectroscopic signatures for intermediate
peptide:metal complexes. Future work will include further
analysis of folding by NMR and CD spectroscopy.
MALDI MASS SPECTROSCOPY
150
Histidine* peptide in nanopure water
125
peptide
Glu
Glu
Glu
Glu
Glu
Glu
Asn
Asn
Asn
Asn
Asn
Asn
Gly
Gly
Gly
Gly
Gly
Gly
Lys
Lys
Lys
Lys
Lys
Lys
His
His
His
His
His
His
Val
Val
Val
Val
Val
Val
Trp
Trp
Trp
Trp
Trp
Trp
Hα Chemical Shift
Intens. [a.u.]
150
75
1640.311
15
100
10
50
5
50
25
0.2
0.1
0
Ser
Trp
Thr
Val
Glu
Asn
Gly
Lys
His
Val
Trp
Arg
-0.1
0
1640
1641
1642
1643
1644
1645
m/z
-0.2
1640.311
peptide + Cu
0
0
750
1000
1250
1500
1750
2000
2250
2500
1560
2750
1570
1580
1590
1600
1610
1620
1630
1640
m/z
EQUILIBRIUM RESTRICTED FACTOR ANALYSIS
OF UV-VIS TITRATION USING SIVVU.ORG
Amino Acid
Conclusions
0.15
0.10
0.05
0.00
200
300
400
500
Wavelength (nm)
600
Absorptivity Curves
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
700
800
Concentration Profiles
60
(1) peptide + Cu ↔ peptide:Cu
(2) peptide:Cu + Cu ↔ peptide:Cu2
∆G°1 = -33.2(4)
∆G°2 = -36.4(6)
RMS Residual: 0.00184145
300
Cu(II)
-0.3
-0.4
UV-Vis titration of 180uM Valine* peptide into 50uM Cu(II)
in 25mM MES buffer from 0 to 2.4 equivalents peptide
data analyzed using sivvu.org
0.20
200
1650
m/z
Raw Data
0.30
Arg
Arg
Arg
Arg
Arg
Arg
0.3
200
Absorbance
His
Asn
Cys
Tyr
Val
Ile
peptide + Na
100
Molar Absorptivity
Thr
Thr
Thr
Thr
Thr
Thr
1610.335
0.4
peptide
0.25
Trp
Trp
Trp
Trp
Trp
Trp
1578.365
0.5
250
Peptide Sequence & Structure
Ser
Ser
Ser
Ser
Ser
Ser
TOCSY NMR OF VALINE* PEPTIDE
Histidine* peptide + Cu(II) in MES buffer
300
Concentration (uM)
0.00
Intens. [a.u.]
Metal Binding
Intens. [a.u.]
Abstract
400
peptide
500
600
Wavelength (nm)
peptide:Cu(II)
700
peptide:Cu(II)2
50
40
30
20
10
0
800
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
• All six peptides adopt β-hairpin structure, stabilized by
aromatic-aromatic interactions
• Copper and Nickel complex to peptide as shown by MALDI
mass spectroscopy
• NMR titration of copper suggests that binding occurs
between residues 4 & 9 in histidine “binding pocket”
• Factor analysis of UV-Vis data shows 1:1 metal to peptide
complexation to be most significant
• The size, polarity, and charge of the amino acid across from
histidine (residue 4) affect binding affinity.
Equivilants Peptide Added
[Cu(II)]
[peptide]
[peptide:Cu(II)]
[peptide:Cu(II)2]
NMR TITRATION OF CU(II) INTO TYROSINE* PEPTIDE
Acknowledgements
• Calvin College Chemistry and Biochemistry Department
• Professor Chad Tatko
• National Science Foundation
NOTE: Amino acids named with a star (*) refer to the peptide
with that amino acid as residue 4 of the sequence