Role in function: The Phenylalanine side chain is fairly nonreactive, and is thus rarely directly involved in protein function, though it can play a role in substrate recognition.
Phenylalanine（苯丙氨酸）
Role in structure: Being hydrophobic, Phenylalanine prefers to be buried in protein hydrophobic cores. The aromatic side chain can also mean that Phenyalanine is involved in stacking interactions with other aromatic side-chains.
紫外/可见光谱在蛋白质研究中的应用

浓度测定
构象变化研究 相互作用研究


蛋白质中氨基酸的紫外吸收光谱
1mM
0.1mM
0.1 肽键在<250 nm的远紫外区有较大吸收
蛋白质构象变化研究
Absorption spectra of Poly-L-Lys HCl: random coil at pH 6.0, 25º C(bold); -Helix at pH 10.8, 25º C(dotted); -strand at pH 10.8, 52º C(dashed).
pH difference spectrum denaturation difference spectrum disturbance difference spectrum
芳环氨基酸的重要性 Tyrosine（酪氨酸）
Role in structure: partially hydrophobic, Tyr prefers to be buried in protein hydrophobic cores. Tyr involved in stacking with other aromatic sidechains. Role in function: Tyr contains a reactive hydroxyl group, thus making it much more likely to be involved in interactions with nonprotein atoms.
is in a non-polar solvent the Trp is on the surface of the protein or conformation changes such that it is brought to
the surface.
Continue
If a quencher (iodide, nitrate ions) quenches Trp fluorescence it must be on the surface of the protein. Failure to do so means its either internal, in a small crevice（裂缝）, or in a highly charged region. Trp fluorescence is quenched by neighbouring protonated acid groups. If the pK measured by monitoring Trp fluorescence is the same as the pK for a known ionizable group (e.g. a carboxyl) then the group must be near the Trp. If a substance that affects the Q of the free amino acid doesn’t affect the fluorescence of the protein it must do so by producing a conformational change. If a substance binds to a protein & Trp fluorescence is quenched, either there is a conformational change as a result of binding or Trp is in or near the binding site.
A common role for Tyr (Thr) within intracellular proteins is phosphorylation. Protein kinases frequently attach phosphates to Tyrosines in order to fascilitate the signal transduction process. Protein kinases are highly specific (i.e. Tyrosine kinases generally do not work on Serines/Threonines and vice versa). Also aromatic amino acids are common in antibody variable domains and at the interface of other protein-protein complexes.
decreases. If the max is shifted to shorter wavelengths when the protein is in a polar solvent, the Trp must be internal.
if max is shifted to shorter wavelengths when the protein
All fluorescence of a protein is due to Trp, Tyr or Phe, unless it contains an extrinsic fluor or fluorescent co-factor. Trp is the dominant intrinsic fluorophore in proteins.
In general
In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids. Aromatic residues can also be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions.
紫外与荧光光谱在蛋白质结构研究 中的应用
1 紫外差光谱法 (UV difference spectrum)
利用分光光度法测定大分子溶液状态下的构象，其原理
是利用环境对生物大分子生色基团的微扰而使吸收峰值的
位置、强度和带宽发生变化，如利用溶剂、pH、温度、 浓度等的微扰，使蛋白分子的酪氨酸的吸收峰发生变化， 从而推断这些生色基团在大分子中的位置和状态。
Tryptophan(色氨酸)
Role in structure: Being hydrophobic, it prefers to be buried in protein hydrophobic cores. The aromatic side chain can also mean that Tryptophan is involved in stacking interactions with other aromatic side-chains. Role in function: As it contains a non-carbon atom (nitrogen) in the aromatic ring system, Tryptophan is more reactive than phenylalanine（苯丙氨酸） though it is less reactive than tyrosine. The Tryptophan nitrogens can play a role in binding to non-protein atoms, but such instances are rare.
Trp fluorescence is very sensitive to its local environment
The max of Trp shifts to shorter wavelengths & the
intensity of max increases as the polarity of the solvent
蛋白质折叠/变性研究
2 荧光的基本原理
荧光是指电子从单线态第一激 发态返回到基态时释放的光。
Overview of Excitation and Emission Fundamentals
ways of measuring fluorescence
Emission spectrum- excitation λ constant, measure fluorescence intensity of emission against λ, I.e. spectrum of emitted light. Excitation spectrum – measure fluorescence intensity at different excitation λ, similar to absorption spectra.