1. Metal-Protein Interactions from the Protein’s Perspective
Supplemental Reading
Prof. Arthur D. Tinoco
University of Puerto Rico, Rio Piedras Campus
Chemistry 8990(013)
Semester

2. 1. Typical Protein Metal Coordination Sites

3. 2. Metal Binding to Proteins can Influence Protein Stability
We 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 performed
Facilitate interaction with a specific receptor for transport or hormone signaling

4. The 4 Levels of Protein Structure

5. Proper Folding Conditions
Denatured Protein
Native Protein Form X
In some cases
Most Stable Form

6. 3. Metal Interactions with Serum Proteins
Serum 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

7. 3A. Human Serum Albumin
kD protein with three homologous helical domains (I-III)
Dominantly -helical
2. Most abundant protein in the blood (600 μM)
3. Binds a variety of ligands and improves their solubility
7 fatty acid (FA) sites
2 drug sites (DS)
4 soft/intermediate metal binding sites
4. Sequestration agent that affects the pharmacokinetics of drugs
Binding extends drug “survival” in the blood

8. 3AI. Metal Binding at the N-terminus of HSA
R1 = Asp
R2 = Ala CH3
N-terminal sequence: DAH
Cu2+
The HSA N-terminus is a Cu2+/Ni2+ 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

9. 3AII. Cu2+ binds with high affinity to Albumin
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,

10. 3AIIa. Isothermal Titration Calorimetry
The 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

11. 3AIIb. Cu(BSA) binding constant determined after accounting for Cu2+ Speciation
Competition Assay:
Note: Tris is a commonly used buffer in biological applications but it is also a metal binder and could affect metal affinity assays.

12. 3AIIb. Cu(BSA) binding constant determined after accounting for Cu2+ Speciation
Competition Assay:
[Cu(BSA)][H+]2
log [H+]2 +
log
[Cu(BSA)]
Kcalc =
log Kcalc =
[Cu2+][BSA]
[Cu2+][BSA]
2 x log [H+] +
log
[Cu(BSA)]
log Kcalc =
At pH 7.4, K?
[Cu2+][BSA]
2 x (-pH) +
log
[Cu(BSA)]
log
[Cu(BSA)]
-1.34 = =
= log KpH 7.4
[Cu2+][BSA]
[Cu2+][BSA]

13. 3B. Human Serum Transferrin (HsTf)
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

14. 3BI. HsTf is a Hard Metal Binder (and Lewis Base)
Binding Site:
2 Tyrosinates
1 Histidine
1 Aspartate
1 Carbonate
Metal affinity (log K1) for OHˉ is correlated with affinity for HsTf (C-lobe).
Li, H.; Sadler, P. J.; Sun, H. Eur J Biochem 1996, 242,

15. 3BII. Fe(III) Binding Alters HsTf Conformation
Change on a tertiary level NI
NII CI
CII
ApoTf
HoloTf

16. 3BII. Fe(III) Binding Alters HsTf Conformation
ApoTf
Metal binding residues:
N1: 1-92 and
N2:
Asp63 (N1)
His249 (N1)
Tyr95 (N2)
Tyr188 (N2) N1 N2

17. 3BII. Fe(III) Binding Alters HsTf Conformation
ApoTf
Metal binding residues:
C1: and
C2:
Asp392 (C1)
Tyr426 (C2)
Tyr517(C2)
His585 (C1) C1 C2

18. 3BIII. Differences in Fe(III) binding to the N-lobe and C-lobe
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-1cm-1 based on [protein]
ε = 2,500 M-1cm-1 based on [Fe(III)]
HoloTf
ApoTf

19. 3BIIIa. Differences in Secondary Sphere of Coordination
H-Bonds between the Arg and CO32-
N-lobe
C-lobe

20. 3BIIIb. The Dilysine Trigger in the N-lobe
Fe(III) Coordination lowers the pKa of K206 and K296
Normal pKa is 10.53, positively charged at pH 7.4
Recall, if pH < pKa, then will be protonated, especially if more than pH unit lower.
pH > pKa, then deprotonated
In the Fe(III) bound closed conformation structure, one of the Lys is deprotonated and the two Lys 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,

21. 3BIIIb. The Dilysine Trigger in the N-lobe
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,

22. 3BIIIb. The Dilysine Trigger in the N-lobe
H-Bonds:
Stronger H-Bond
Donor
Acceptor
Linear
Bent
Donor-Acceptor Distances (Å)
Relative Strength
Bond Strength (kcal/mol)
2.2 – 2.5
Strong, mostly covalent
40-14
Moderate, mostly electrostatic
15-4
Weak
<4
Bond lengths can be a little misleading if H-bond is bent.

23. 3BIIIb. The Dilysine Trigger in the N-lobe
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: /srep00999

24. 3BIIIc. The pH sensitive triad in the C-lobe
Fe(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,

25. 3BIIId. Differences in the Stability of Fe(III) Bound C and N-lobe
There 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 .
Transition
Folded Protein
Denatured Protein

26. 3BIIId. Differences in the Stability of Fe(III) Bound C and N-lobe
Differential scanning calorimetry, a thermal application that uses heat measurements to characterize protein denaturation, was applied to Fe2-HsTf
N-lobe
C-lobe
Tm =68.4 °C
Tm =57.6 °C
Lin, L-N. et al. Biochemistry. 1994, 33,

27. * 2nd Fe(III) * 1st Fe(III)
First Fe(III) binds to the C-lobe (higher affinity) and this increases the Tm of that lobe to 87 °C.
Δ Tm (C-lobe) = 29.4 °C (HUGE!!!!)
The N-lobe Tm also shifts upward by ~5 °C due to cooperativity
Second Fe(III) binds to the N-lobe and this increases the Tm of that lobe to 87 °C.
Δ Tm (N-lobe) = 18.6 °C (NOT TOO SHABBY EITHER!!!)
2nd Fe(III) *
1st Fe(III)

28. Take Home Message STABILITY INCREASE
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

29. Gaining Access to Protein Crystal Structures
Protein data bank (

30. Download and save the pdb file.

31. Use Pymol to Visualize the Structures via the PDB files

32. For more information about a protein go to www.uniprot.org
Gives protein sequence information
-Indicates which residues are cleaved after being synthesized
Metal binding site residues
Glycosylation sites