Joao H C, Dwek R A
Department of Biochemistry, University of Oxford, England.
Eur J Biochem. 1993 Nov 15;218(1):239-44. doi: 10.1111/j.1432-1033.1993.tb18370.x.
In an attempt to elucidate the role of carbohydrates on protein structure and dynamics in glycoproteins, ribonuclease B (RNase B), containing a single glycosylation site at Asn34, has been investigated and compared with the enzyme in the unglycosylated form (RNase A). RNase B consists of five glycoforms: Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2 (Man5-9GlcNAc2). The individual glycoforms Man1GlcNAc2 (synthetic) and Man5GlcNAc2 of RNase B have been studied to ascertain any specific effects of the different oligosaccharides. NMR measurement of amide-proton/deuterium exchange rates shows that glycosylation of the enzyme leads to the protection of amide-proton resonances from solvent exchange for a large number of residues, both in the vicinity of the glycosylation site (e.g. residues 29-34 and 35) and remote from it (e.g. residues 57-61 and 75-76). In addition, residues 10-13, 32, 34 and 35, which are observed to be protected from exchange as a result of glycosylation in the individual glycoforms Man1Glc-NAc2-RNase and Man5GlcNAc2-RNase (when compared with RNase A) are less protected in RNase B. This additional protection in the glycoforms Man1GlcNAc2-RNase and Man5GlcNAc2-RNase may arise from steric hindrance between the oligosaccharide and protein reducing solvent accessibility. The rates of solvent exchange of amide protons for residues 10-13, 32, 34 and 35 are dependent on the oligosaccharide moiety. The average amide-proton/deuterium exchange rate in Man6-9Glc-NAc2-ribonucleases for residues 10-13 and 35 is approximately three times greater than Man5Glc-NAc2-ribonuclease, while for residues 32 and 34 it is approximately 7-11 times greater. CD analysis of RNase A and RNase B revealed the carbohydrate moiety to have a small stabilizing effect (approximately 5 kJ/mol) on the protein.
为了阐明碳水化合物在糖蛋白中对蛋白质结构和动力学的作用，对在Asn34处含有单个糖基化位点的核糖核酸酶B（RNase B）进行了研究，并与未糖基化形式的酶（RNase A）进行了比较。RNase B由五种糖型组成：Man5GlcNAc2、Man6GlcNAc2、Man7GlcNAc2、Man8GlcNAc2和Man9GlcNAc2（Man5 - 9GlcNAc2）。已对RNase B的单个糖型Man1GlcNAc2（合成的）和Man5GlcNAc2进行了研究，以确定不同寡糖的任何特定作用。酰胺质子/氘交换率的核磁共振测量表明，该酶的糖基化导致大量残基的酰胺质子共振免受溶剂交换的影响，这些残基既在糖基化位点附近（例如残基29 - 34和35），也远离该位点（例如残基57 - 61和75 - 76）。此外，在单个糖型Man1Glc - NAc2 - RNase和Man5GlcNAc2 - RNase中（与RNase A相比），由于糖基化而免受交换影响的残基10 - 13、32、34和35在RNase B中受到的保护较少。在糖型Man1GlcNAc2 - RNase和Man5GlcNAc2 - RNase中的这种额外保护可能源于寡糖与蛋白质之间的空间位阻，降低了溶剂可及性。残基10 - 回首页13、32、34和35的酰胺质子的溶剂交换速率取决于寡糖部分。对于残基10 - 13和35来说，Man6 - 9Glc - NAc2 - 核糖核酸酶中的平均酰胺质子/氘交换率大约是Man5Glc - NAc2 - 核糖核酸酶的三倍，而对于残基32和34，它大约是7 - 11倍。RNase A和RNase B的圆二色性分析表明，碳水化合物部分对蛋白质具有较小的稳定作用（约5 kJ/mol）。
