Spectroscopic tools for improved protein secondary structure analysis in protein-heparan sulfate complexes

许多生物过程，如细胞生长、分裂和对体内条件的反应，都依赖于蛋白质和细胞表面配体之间的相互作用。检测这些相互作用并了解当蛋白质与靶配体结合时发生的变化在技术上具有挑战性。然而，这些信息是至关重要的，不仅是为了更好地理解这个过程，而且如果我们能够控制它们或创造新的治疗方法。对于一组相互作用，即蛋白质和细胞表面受体硫酸肝素（HS）之间的相互作用，这个问题更加明显。这是因为目前基于圆二色性（CD）和红外（FTIR）光谱的技术不能很好地处理HS， HS显示出干扰复合物蛋白质成分分析的信号。我们建议探索两种方法，这两种方法可能使我们能够选择性地探测HS存在下蛋白质的结构变化。第一个是基于从我们现有的结构库中选择一个活跃的但结构上修改的HS片段。这将被选择，使其没有特定的群体，不会在红外（FTIR）中出现干扰信号，但将保持生物活性。我们称之为“透明”HS配体。利用这种方法，当检测到蛋白质的信号时，HS的信号应该消失。结构变化应该只从蛋白质中报告。第二种方法利用了一种叫做振动圆二色性（VCD）的技术，它可以检测到蛋白质中存在的规则结构，但不能检测到结构不那么规则的HS配体（一种正常的，而不是修饰的HS配体）。这可能使该蛋白即使在HS存在下也能被选择性地观察到。拟议的研究是用两种已知在结合HS时改变形状的蛋白质来测试这两种可能性。一种是朊病毒蛋白（PrPC）的正常（非传染性）形式，另一种是参与阿尔茨海默病斑块形成的酶；BACE-1。如果成功，该方法将广泛适用于研究蛋白质与细胞表面HS配体之间发生的大量蛋白质-HS相互作用，这些相互作用目前无法精确跟踪。

Many biological processes such as cell growth, division and responses to conditions in the body depend on interactions between proteins and cell-surface ligands. It is technically challenging to detect these interactions and understand what changes are occurring in the proteins when they bind their target ligand. However, such information is vital, not only for a better understanding of the process, but also if we are to be able to control them or to create new treatments. This problem is even more stark for one set of interactions, those between proteins and the cell-surface receptor heparan sulfate (HS). This is because the current techniques, based on circular dichroism (CD) and infra-red (FTIR) spectroscopy do not cope well with HS, which exhibits signals that interfere with the analysis of the protein component of the complex. We propose to explore two approaches which may enable us to selectively probe the structural changes in proteins in the presence of HS. The first is based on selection of an active but structurally modifed HS fragment from our existing library of structures. This will be selected so that it lacks particular groups and will present no interferring signal in infra-red (FTIR) but will retain biological activity. This we term a 'transparent' HS ligand. Using this, the signal for HS should disappear while that of the protein is detected. Structural changes should then only be reported from the protein. The second method utilises a technique called vibrational circular dichroism (VCD), which should detect the regular structures present in the protein but not those of the less-regularly structured HS ligand (a normal, not a modified HS ligand). This may enable the protein to be selectively observed even in the presence of HS. The proposed research is to test these two possibilities with two proteins that are known to change shape on binding HS. One is the normal (non-infectious) form of the prion protein (PrPC) and the other is an enzyme involved in plaque formation in Alzheimers' disease; BACE-1. If successful, the approach will be widely applicable to studying the great number of protein-HS interactions that occur between protein and cell-surface HS ligands, which currently cannot be accurately followed.