Luo Y, Kay M S, Baldwin R L
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
Nat Struct Biol. 1997 Nov;4(11):925-30. doi: 10.1038/nsb1197-925.
The apomyoglobin pH 4 folding intermediate contains the A, G, and H helices of myoglobin. Helix destabilizing mutations in the A and G helices are used to test whether the pH 4 folding intermediate of apomyoglobin folds cooperatively. Single glycine or proline mutations destabilize the intermediate substantially, showing that intrinsic helix propensities are important for stability of the intermediate. The A and G helices interact to stabilize each other, as shown by the effect of mutations in the G helix on the unfolding of the A helix, which can be monitored by tryptophan fluorescence. Wild type and the most stable mutant unfold in a two-state reaction, as shown by superposition of the unfolding curves measured by two probes (far-UV circular dichroism and Trp fluorescence), while unfolding of the less stable mutants is more complex. Cooperativity and stability of folding are linked also when stabilizing anions (sulphate, perchlorate) are used to adjust stability.
脱辅基肌红蛋白pH 4折叠中间体包含肌红蛋白的A、G和H螺旋。利用A和G螺旋中的螺旋去稳定化突变来测试脱辅基肌红蛋白的pH 4折叠中间体是否协同折叠。单个甘氨酸或脯氨酸突变会显著破坏中间体的稳定性，表明内在螺旋倾向对中间体的稳定性很重要。A和G螺旋相互作用以彼此稳定，如G螺旋中的突变对A螺旋解折叠的影响所示，这可以通过色氨酸荧光监测。野生型和最稳定的突变体以两态反应解折叠，如通过两种探针（远紫外圆二色性和色氨酸荧光）测量的解折叠曲线叠加所示，而稳定性较差的突变体的解折叠则更为复杂。当使用稳定阴离子（硫酸盐、高氯酸盐）来调节稳定性时，折叠的协同性和稳定性也有关联。
