University of Puerto Rico Rio Piedras Campus Chemistry 8990013 Semester 1 20142015 1 MetalProtein Interactions from the Proteins Perspective Supplemental Reading 1 Typical Protein Metal Coordination Sites ID: 780116
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Slide1
Prof. Arthur D. TinocoUniversity of Puerto Rico, Rio Piedras CampusChemistry 8990(013)Semester 1 2014-2015
1
Metal-Protein Interactions from
the Protein’s Perspective
Supplemental Reading
Slide21. Typical Protein Metal Coordination Sites 2
Slide32. Metal Binding to Proteins can Influence Protein Stability3We have explored how biomolecular chelation of metals can increase both metal solubility, stability, and bioavailability. It can also have an impact on the structure, stability, and function of the biomolecule. Metal binding can:
Change the conformation of a biomolecule and improve its stability
Enable a particular function to be performedFacilitate interaction with a specific receptor for transport or hormone signaling
Slide4The 4 Levels of Protein Structure4
Slide55
Most Stable Form
Denatured Protein
Native Protein Form
Proper Folding Conditions
X
In some cases
Slide63. Metal Interactions with Serum Proteins6Serum proteins are great case studies for investigating the impact that metal binding has on protein stability because they are typically the primary vehicles for distribution of metals throughout our body.Two of the major proteins for metal transport:Human Serum Albumin (HSA)- An example where metal binding has no effect on global protein structure
Human Serum Transferrin (
HsTf)- An example where metal binding results in significant conformational change
Slide73A. Human Serum Albumin7
1. 66.5
kD
protein
with three homologous
helical
domains (I-III
)
Dominantly
-helical
2
.
Most abundant
protein
in
the blood
(600
μ
M)
3
. Binds
a variety of
ligands and improves
their solubility
7 fatty acid (FA) sites
2 drug sites (DS)
4 soft/intermediate
metal binding sites4. Sequestration agent that affects the pharmacokinetics of drugsBinding extends drug “survival” in the blood
Slide8Cu
2+
3
AI. Metal Binding at the N-terminus of HSA
8
The HSA N-terminus is a Cu
2+
/Ni
2+
binding site:
In the
apo
form (metal unbound), highly unstructured unlike the majority of the protein.
In the
holo
form (metal bound), the sequence is configured as an intermediate metal binder
The protein secondary and tertiary structure remain unchanged as indicated by circular
dichroism
and fluorescence
R
1
= Asp
R
2
=
Ala
-CH
3
N-terminal sequence: DAH
Slide93AII. Cu2+ binds with high affinity to Albumin
9
Wilcox (2002) studied Cu2+ binding to bovine SA (BSA) using isothermal titration
calorimetry.
Zhang, Y. and Wilcox, D.E.
J. Biol.
Inorg
. Chem.
2002
,
7
, 327-337.
Slide103AIIa. Isothermal Titration Calorimetry10The technique measures the energy associated with a chemical reaction triggered by the mixing of two components.Involves the stepwise addition of one reactant (typically metal) into the reaction cell containing
the other reactant (protein)Energy is either released (bond
forming) or absorbed (bond breaking) as metal binds to
the protein
Slide113AIIb. Cu(BSA) binding constant determined after accounting for Cu2+ Speciation11Note: Tris is a commonly used buffer in biological applications but it is also a metal binder and could affect metal affinity assays.
Competition Assay:
Slide123AIIb. Cu(BSA) binding constant determined after accounting for Cu2+ Speciation 12
Competition Assay:
K
calc
=
[Cu(BSA)][H
+
]
2
[
Cu
2+
][BSA]
log
K
calc
=
[Cu(BSA
)]
[
Cu
2+
][BSA]
log [H
+
]
2
+
log
log
K
calc
=
[Cu(BSA
)]
[
Cu
2+
][BSA]
2 x log [H
+
] +
log
At pH 7.4, K?
-1.34 =
[Cu(BSA
)]
[
Cu
2+
][BSA]
2 x (-pH) +
log
13.46 =
[Cu(BSA
)]
[
Cu
2+
][BSA]
log
= log
K
pH
7.4
Slide133B. Human Serum Transferrin (HsTf)13
1.
A member of the transferrin family of
proteins known to be either
monolobal
(~
40
kD
)
or
bilobal
(~ 80
kD
)
Proposed ancient gene duplication may
have resulted in
bilobal
transferrin
~40% sequence homology between lobes
2. Multiple Functions
Iron transport/
homeostatis
Bacteriostasis
- Bacteria thrive on iron and a strong
chelator
prevents them from having access
to it.
3. Binds Hard Metals
Fe-Tf C lobe: log K = 22.2 Fe-Tf N lobe: log K = 21.3
Slide143BI. HsTf is a Hard Metal Binder (and Lewis Base)14Metal affinity (log K1) for OH
ˉ is correlated with affinity for
HsTf (C-lobe).
Li, H.; Sadler, P. J.; Sun, H. Eur
J
Biochem
1996,
242
, 387-393.
Binding Site
:
2
Tyrosinates
1 Histidine
1 Aspartate
1 Carbonate
Slide153BII. Fe(III) Binding Alters HsTf Conformation15NI
NIICI
CII
ApoTf
HoloTf
Change on a tertiary level
Slide163BII. Fe(III) Binding Alters HsTf Conformation16
Metal binding residues:
N1: 1-92 and 247-330
N2:
93-246
Asp63 (N1)
His249 (N1
)
Tyr95 (N2)
Tyr188 (N2)
ApoTf
N1
N2
Slide173BII. Fe(III) Binding Alters HsTf Conformation17
Metal binding residues:
C1: 340-425 and 573-679
C2: 426-572
Asp392 (C1)
Tyr426 (C2)
Tyr517(C2)
His585 (C1)
ApoTf
C
1
C
2
Slide183BIII. Differences in Fe(III) binding to the N-lobe and C-lobe18
A difference is not detectable by UV-Vis
LMCT band (470) produces characteristic pink color when Fe(III) binds to
HsTf.
The increase in absorbance due to Fe(III) binding to the two sites is comparable.
ε = 5,000 M
-1
cm
-1
based on [protein]
ε
=
2,500
M
-1
cm
-1
based on
[Fe(III)]
HoloTf
ApoTf
Slide193BIIIa. Differences in Secondary Sphere of Coordination19N-lobe
C-lobe
H-Bonds between the
Arg
and CO
3
2-
Slide203BIIIb. The Dilysine Trigger in the N-lobe20Fe(III) Coordination lowers the pKa of K206 and K296Normal pKa is 10.53, positively charged at pH 7.4
Recall, if pH < pKa, then will be protonated, especially if more than
1 pH unit lower. pH >
pKa, then deprotonated
In the Fe(III) bound closed conformation structure, one of the Lys is deprotonated and the two
L
ys residues engage in H-Bond via a single H+
This hydrogen bond interaction is stable even at pH 5.5.
Gumerov
, D.R. and
Kaltashov
, I.A.
Anal Chem.
2001
,
73
, 2565-2570.
Slide213BIIIb. The Dilysine Trigger in the N-lobe21 The H-Bond is stabilized by decreased exposure to solvent and the hydrophobic box created by Y188, Y95, and H249, which favors lower charge.
Halbrooks, P.J. et al
. Biochemistry.
2005, 44, 15451-15460.
Slide223BIIIb. The Dilysine Trigger in the N-lobe22H-Bonds:
Linear
Bent
Stronger H-Bond
Donor
Acceptor
Donor-Acceptor
Distances (Å)
Relative Strength
Bond Strength (kcal/
mol
)
2.2 – 2.5
Strong, mostly
covalent
40-14
2.5-3.2
Moderate, mostly electrostatic
15-4
3.2-4.0
Weak
<4
Bond lengths can be a little misleading if H-bond is bent.
Slide233BIIIb. The
Dilysine Trigger in the N-lobe
23
Any sudden influx in protons would disrupt this interaction and lead to
the “trigger” of conformational change
Closed Open
It used to be thought that an
open
conformation
meant that Fe(III)
release
would
occur.
However, a
recent crystal
structure study suggests
that binding of
additional synergistic
anions can lead to
an Fe(III
) bound, open
conformation
state
.
Yang, N.
et al.
Sci. Rep.
2012
, DOI:10.1038/srep00999
Slide243BIIIc. The pH sensitive triad in the C-lobe24Fe(III) Coordination lowers the pKa of K534 and R632 and results in possible extensive H-Bond network with each other and D634. This H-Bond network stable even at pH 5.5 due to YYH hydrophobic box. There are also extensive elextrostatic interactions involved.
Halbrooks
, P.J.
et al
.
Biochemistry
.
2005
,
44
, 15451-15460.
Slide253BIIId. Differences in the Stability of Fe(III) Bound C and N-lobe25There are numerous ways to measure the stability of a protein and it is often done by using either a chemical or thermal method to examine the transition from a folded to unfolded (denatured) state. These methods can be used to examine stability differences between different protein conformations .
Folded Protein
Denatured Protein
Transition
Slide263BIIId. Differences in the Stability of Fe(III) Bound C and N-lobe26Differential scanning calorimetry, a thermal application that uses heat measurements to characterize protein denaturation, was applied to Fe2-HsTf
C-lobe
N
-lobe
T
m
=57.6 °C
T
m
=68.4 °C
Lin, L-N.
et al
.
Biochemistry
.
1994
,
33
, 1881-1888.
Slide27271st Fe(III) First Fe(III) binds to the C-lobe
(higher affinity) and this increases the Tm
of that lobe to 87 °C.
Δ
T
m
(C-lobe)
= 29.4
°
C
(HUGE!!!!)
The N-lobe T
m
also shifts upward by ~5
°
C due to
cooperativity
Second
Fe(III) binds to the
N-lobe
and
this increases
the
T
m
of that
lobe to 87 °C. Δ Tm (N-lobe) = 18.6 °
C (NOT TOO SHABBY EITHER!!!) *2nd Fe(III)*
Slide28Take Home Message28 Compactness of a globular protein, Stability
In the case of transferrin, in going from
apo
to
holo
form, you are essentially transitioning to a more stable protein form. This is particularly true if you increase intramolecular contacts.
STABILITY INCREASE
Slide2929Gaining Access to Protein Crystal StructuresProtein data bank (www.rcsb.org):
Slide3030Download and save the pdb file.
Slide3131Use Pymol to Visualize the Structures via the PDB files
Slide3232For more information about a protein go towww.uniprot.orgGives protein sequence information-Indicates which residues are cleaved after being synthesizedMetal binding site residues
Glycosylation sites