趨化素 (chemokine) 是一種具有吸引白血球的能力以及抑制或是促進血管新生的功能的蛋白，它的各種聚合狀態 (oligomerization state) 和它能影響的生物功能息息相關。Platelet factor-4 (PF4又稱作第四型血小板因子、CXCL4) 為第一個被發現具有抑制血管新生的趨化素。在過去的研究中指出，四聚體的PF4和黏多醣 (GAGs) 具有很強的結合能力，而單體的PF4也被報導說可能和膜蛋白受體(GPCR)結合的能力並進行其下游的反應。趨化素能以不同型態的多聚體來執行不同的的生理活性，研究它如何形成聚合狀態的因素可以使我們能精準地調控下游所引發的反應。然而在過去有關於趨化素聚合狀態的研究，多聚體的形成和電荷-電荷的交互作用力息息相關，利用改變溶液的酸鹼度和氯化鈉的含量使得胺基酸改變其電性或破壞其鹽橋作用來使得四聚體的PF4形成二聚體。另外對於界面上的胺基酸也利用了點突變來改變界面的電性，使得本來相吸的交互作用力改為相斥，以期望得到穩定的PF4二聚體。我們進行了改變溶液酸鹼度及鹽類含量利用核磁共振的二維光譜觀察是否在PF4上可以改變它的多聚體型態；也對點突變後的PF4進行了顆粒大小排阻(size-exclusion)、超高速離心分析、肝素 (heparin) 的結合實驗和核磁共振二維光譜來觀察其生物物理特性與多聚體型態的改變。我們發現了PF4 在不同的酸鹼度和鹽類含量下可以改變其多聚體型態與結構；其他點突變的PF4也對其結構和多聚體型態與結構造成影響，因此我們認為界面上的胺基酸帶電性對於整個多聚體的形成非常重要。未來可以利用其他點突變的胺基酸來進行更深入的研究，期望得到穩定的各種型態的PF4。

Chemokine is a protein that is described for their abilities to recruit leukocytes and affect inflammatory. Chemokines has different oligomerization states to execute different bio-functions. Platelet factor-4 (PF4 or named as CXCL4) is the first chemokine described as anti-angiogenesis chemokine. Tetrameric PF4 has high affinity to glycosaminoglycans (GAGs) while monomeric PF4 has been speculated to bind with G-protein coupled receptor (GPCR) to trigger the downstream signal. For execute the bio-functions, PF4 oligomers should be precisely controlled and the factor in mediating PF4 assembling is worthwhile for studying. There is a reported relationship between charge-charge interaction and oligomerization states of chemokines. By changing the content of salt and pH value, PF4 has distinct salt-bridge interaction and protonation status of residues. The effects modulate the transition of tetramer, dimer and monomer. We made point mutations on the interface to disrupt the intrinsic interaction. NMR HSQC method reported PF4 basic structural property and oligomerization states. We also tested the experiments of heparin binding, size-exclusion chromatography and analytical ultracentrifuge to classify the biophysical properties. We reveal that charge-charge and salt-bridge interaction actually dominantly regulates the oligomerization states, as the PF4 mutations with influence in structure and oligomerization states. In the future, we could do deeper research on the factors of oligomerization states by other point mutations. The effort would contribute in the preparation of pure oligomerization states of PF4.

Contents
Abstract 2
中文摘要 3
Abbreviations 6
1. Introduction 7
1.1 Chemokine 7
1.2 Structure and Oligomerization state of PF4 7
1.3 Angiogenesis 8
1.4 Functional active formation of PF4 9
1.5 Charge-Charge interaction in PF4 tetramer interface 9
1.6 Aim of Study 11
2. Materials and Methods 21
2.1 Point Mutations K50E, S26E, S26E/K50E 21
2.3 Protein purification 22
2.4 Preparation 15N-labeled sample for NMR 23
2.5 Analytical ultracentrifuge 23
2.6 HSQC experiment 23
2.7 Circular Dichroism Spectroscopy 23
3. Results 26
3.1 PF4 structures under various pH values and ionic strengths. 26
3.2 Biophysical properties of PF4 mutations 27
3.2.1 Mutation site selection 27
3.2.2 Binding affinity in different mutation 27
3.2.3 Size exclusion by gel filtration 28
3.3 Circular dichroism (CD) 29
3.4 NMR study of PF4-S26E and PF4-K50E 29
3.4.1 PF4-S26E 30
3.4.2 PF4-K50E 30
4. Discussion 49
5. References 56

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