靜電荷的交互作用在許多蛋白質與配體結合上扮演著主要的趨力。例如
生長因子與細胞表面之醣氨聚多醣以及脫氧核醣核酸與蛋白的交互作用。自由
能差與HSQC所量測到的化學位移是兩個重要的物理參數常被用來評估特定胺
基酸對於蛋白與配體結合作用的貢獻。然而，從先前的研究發現，自由能差與
HSQC的化學位移一致性並不好。原因在於核磁共振光譜所觀察到的化學位移
是來自與蛋白質主鏈上的變化，然而，主鏈上的變化可能無法充分的反應發生
在長支鏈尾端（例如Arginine, Lysine）的交互作用。基於偵測胺基酸支鏈會比
主鏈叫為靈敏的假設下，本研究使用核磁共振儀H2CN的實驗，來激發
Lysine/Arginine氨基酸支鏈上無法交換的氫原子，再藉由選取適當的耦合係數
（J-coupling constant）將磁化訊號傳遞至以化學鍵相接的碳及氮原子上，來偵
測藉由靜電荷交互作用與蛋白質結合的配體。藉此偵測蛋白質胺基酸支鏈的實
驗，本論文確實證實能得到相較於傳統HSQC實驗偵測蛋白質主鏈更確切的結
合資訊。

Electrostatic interaction is a major driving force for several cases of protein
binding to ligand, such as growth factor to cell surface glycosaminoglycan
and DNA-binding protein to DNA. The free energy difference and
1H-15N HSQC perturbation of protein upon ligand titration are two critical
physical quantities frequently employed for evaluating the binding contribution
of a particular residue. However, based on the results shown in the
previous studies, less correlation was found between the binding free energies
and perturbed HSQC chemical shifts for those residues involved in
charge-charge interactions. The reason might be due to that the changes
observed in 1H-15N HSQC spectra, revealing the protein backbone resonances,
could not represent the interactions occurring in the end of long side
chain, especially for the Lys and Arg long side chain moieties.
In order to clarify the discrepancy, we first utilize the NMR pulse sequence
H2CN, providing information available for the realistic events arising
from amino acid side chains rather than protein backbone by using conventional
HSQC titration experiments, to probe the charge-charge interactions
through the non-exchangeable protons in lysine residues binding to
the counterion ligands. In the present study, we choose hepatoma-derived
growth factor (HDGF) and basic fibroblast growth factor (FGF2) as the protein
models, which possess 70% and 30% heparin binding affinities contributed
from electrostatic interactions, respectively. Our data indicate that in
the H2CN titration experiments, most of chemical shift perturbations occurring
in lysine/argine side chains show a good correlation to the free energy
changes of single-Lys/Arg mutants for both HDGF and bFGF, suggesting
that NMR H2CN provides a precise and quick evaluation of electrostatic interactions
in heparin-binding proteins.
3

[1] I Andre, S Linse, and F A Mulder. Residue-specific pKa determination of
lysine and arginine side chains by indirect 15N and 13C NMR spectroscopy:
application to apo calmodulin. J Am Chem Soc, 129(51):15805–15813,
2007.
[2] Anne Imberty, Hugues Lortat-Jacob, and Serge P´erez. Structural view of glycosaminoglycanprotein
interactions. Carbohydrate Research, 342(34):430–
439, 2007.
[3] Mike PWilliamson. Using chemical shift perturbation to characterise ligand
binding. Progress in Nuclear Magnetic Resonance Spectroscopy, 73(0):1–
16, 2013.
[4] Julie D Forman-Kay, G Marius Clore, and Angela M Gronenborn. Relationship
between electrostatics and redox function in human thioredoxin:
characterization of pH titration shifts using two-dimensional homo- and heteronuclear
NMR. Biochemistry, 31(13):3442–3452, 1992.
[5] J Song, MLaskowski Jr., MA Qasim, and J L Markley. Two conformational
states of Turkey ovomucoid third domain at low pH: three-dimensional structures,
internal dynamics, and interconversion kinetics and thermodynamics.
Biochemistry, 42(21):6380–6391, 2003.