Tominaga N, Jameson D M, Uyeda K
Research Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
Protein Sci. 1994 Aug;3(8):1245-52. doi: 10.1002/pro.5560030810.
Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.
通过跟踪酶活性以及使用荧光方法（强度、发射最大值、极化和猝灭），利用内在（色氨酸）和外在（5-((2-(碘乙酰基)氨基)乙基)萘-1-磺酸）探针，监测大鼠睾丸果糖6-磷酸2-激酶：果糖2,6-二磷酸酶在盐酸胍中的可逆去折叠。去折叠反应最少可描述为从折叠二聚体→部分去折叠二聚体→单体→去折叠单体的四态转变。由于激酶结构域的优先去折叠，部分去折叠的二聚体具有高磷酸酶/激酶比。复性反应通过去折叠单体非常快速地转化（小于1秒）为二聚体进行，此时没有任何酶活性，随后是缓慢（超过60分钟）形成活性酶。激酶和磷酸酶的恢复率相似。因此，重折叠似乎是涉及不同形式瞬时二聚体中间体的去折叠途径的逆转。使用碘化物和丙烯酰胺的荧光猝灭研究表明，包括N端肽中的Trp-15在内的色氨酸仅略微可被碘化物接近，但对丙烯酰胺更易接近。果糖6-磷酸而非ATP或果糖2,6-二磷酸减少了碘化物猝灭，但所有这些配体均抑制丙烯酰胺猝灭25%。这些结果表明，N端肽（含有一个色氨酸）未暴露在蛋白质表面，可能在保护其他色氨酸免受溶剂影响方面发挥重要作用。
