Proteins can also be denatured by extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or by detergents. Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic aggregation of nonpolar amino acid side chains that produces the stable core of globular proteins; urea also disrupts hydrogen bonds; and extremes of pH alter the net charge on a protein, causing electrostatic repulsion and the disruption of some hydrogen bonding.

极端 pH、某些可与水混溶的有机溶剂（如乙醇或丙酮）、某些溶质（如尿素和盐酸胍）以及去污剂，也都能使蛋白质变性。这些变性剂都属于相对温和的处理，因为它们不断裂多肽链中的任何共价键。有机溶剂、尿素和去污剂主要破坏非极性氨基酸侧链的疏水聚集，而这种聚集正是球状蛋白稳定内核的来源；尿素还破坏氢键；极端 pH 则改变蛋白质的净电荷，引起静电排斥并破坏部分氢键。

Structural biology is the study of the three-dimensional structures of biomolecules, including proteins, nucleic acids, lipid membranes, and oligosaccharides. Structural biologists combine biochemical approaches with physical tools and computational methods to obtain these structures. Structural biology is extraordinarily powerful for elucidating the relationships between the structure and function of proteins, the molecular basis for enzymatic catalysis and ligand binding, and evolutionary relationships between proteins. Here we focus primarily on three commonly used methods in structural biology: x-ray crystallography, nuclear magnetic resonance (NMR), and cryoelectron microscopy (cryo-EM). Which method a structural biologist uses depends on the system being studied and what information is to be learned. Often structural biologists combine multiple methods to provide a more complete view of function.

结构生物学研究生物分子的三维结构，对象包括蛋白质、核酸、脂膜和寡糖。结构生物学家把生化手段与物理工具、计算方法结合起来，以获得这些结构。在阐明蛋白质结构与功能的关系、酶催化与配体结合的分子基础以及蛋白质之间的进化关系上，结构生物学极为有力。这里我们主要介绍结构生物学中三种常用方法：X 射线晶体学、核磁共振（NMR）和冷冻电子显微术（cryo-EM）。结构生物学家选用哪种方法，取决于所研究的体系以及想获得什么信息。他们还常常联用多种方法，以更完整地认识功能。

The term secondary structure refers to any chosen segment of a polypeptide chain and describes the local spatial arrangement of its main-chain atoms, without regard to the positioning of its side chains or its relationship to other segments. A regular secondary structure occurs when each dihedral angle, ϕ and ψ, remains the same or nearly the same throughout the segment.

二级结构这一术语针对多肽链中任意选定的一段，描述该段主链原子的局部空间排布，既不涉及其侧链的定位，也不涉及它与其他肽段的关系。当一段肽链中每个二面角 ϕ 和 ψ 都保持相同或近乎相同时，就形成规则的二级结构。

The hydrophobic effect is clearly important in stabilizing conformation; the interior of a structured protein is generally a densely packed core of hydrophobic amino acid side chains. It is also important that any polar or charged groups in the protein interior have suitable partners for hydrogen bonding or ionic interactions.

疏水效应对稳定构象显然很重要；结构化蛋白质的内部，一般是疏水氨基酸侧链紧密堆积而成的内核。同样重要的是，位于蛋白质内部的任何极性基团或带电基团，都必须有合适的配对者来形成氢键或离子相互作用。

The overall three-dimensional arrangement of all atoms in a protein is referred to as the protein’s tertiary structure. Whereas the term “secondary structure” refers to the spatial arrangement of amino acid residues that are adjacent in a segment of a polypeptide, tertiary structure includes longer-range aspects of amino acid sequence.

蛋白质中全部原子的总体三维排布，称为该蛋白质的三级结构。「二级结构」一词指多肽链某一段中彼此相邻的氨基酸残基的空间排布；三级结构则涵盖氨基酸序列上更长程的方面。

Amino acids that are far apart in the polypeptide sequence and are in different types of secondary structure may interact within the completely folded structure of a protein. Interacting segments of polypeptide chains are held in their characteristic tertiary positions by several kinds of weak interactions (and sometimes by covalent bonds such as disulfide cross-links) between the segments. Some proteins contain two or more separate polypeptide chains, or subunits, which may be identical or different. The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure.

在多肽序列上相距很远、又处于不同类型二级结构中的氨基酸，可以在蛋白质完全折叠后的结构里发生相互作用。多肽链中互相作用的各段，靠若干种弱相互作用（有时还靠二硫交联这类共价键）维持在各自特有的三级结构位置上。有些蛋白质含两条或多条独立的多肽链，即亚基，它们可以相同也可以不同。这些亚基在三维复合物中的排布方式，构成四级结构。

a recognizable folding pattern involving two or more elements of secondary structure and the connection(s) between them. A motif can be very simple, such as two elements of secondary structure folded against each other, and may represent only a small part of a protein. An example is a β-α-β loop (Fig. 4-16a). A motif can also be a very elaborate structure involving scores of protein segments folded together, such as the β barrel (Fig. 4-16b). In some cases, a single large motif may comprise the entire protein.

（模体或折叠就是）一种可辨认的折叠模式，由两个或多个二级结构元件以及它们之间的连接构成。模体可以很简单，例如两个二级结构元件互相折叠靠在一起，只占蛋白质的一小部分，β-α-β 环即是一例（图 4-16a）。模体也可以是很复杂的结构，由几十段肽链折叠在一起，例如 β 桶（图 4-16b）。在某些情况下，单个大模体就构成整个蛋白质。

The number of folding patterns is not infinite. Among the tens of thousands of distinct protein structures archived in the PDB, only about 1,400 different folds or motifs are classified by the SCOP2 database. Given the many years of progress in structural biology, new motifs are now discovered only rarely. Many examples of recurring domain or motif structures are available, and these reveal that protein tertiary structure is more reliably conserved than amino acid sequence.

折叠模式的种类并非无限。在 PDB 收录的数万个不同蛋白质结构中，SCOP2 数据库只归类出约 1400 种不同的折叠或模体。经过结构生物学多年的进展，如今很少再发现新的模体。反复出现的结构域或模体结构已有大量实例，它们表明蛋白质的三级结构比氨基酸序列保守得更可靠。

The complex structures of globular proteins can be analyzed by examination of folding patterns, called motifs or folds. The many thousands of known protein structures are generally assembled from a repertoire of only a few hundred motifs. Domains are regions of a polypeptide chain that can fold stably and independently.

球状蛋白质的复杂结构，可以通过考察折叠模式来分析，这些折叠模式称为模体或折叠。已知的成千上万个蛋白质结构，一般都由仅仅几百种模体组装而成。结构域则是多肽链中能够稳定而独立地折叠的区域。

The second term for describing structural patterns is domain. A domain, as defined by Jane Richardson in 1981, is a part of a polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein.

描述结构模式的第二个术语是结构域。按 Jane Richardson 1981 年的定义，结构域是多肽链中的一部分，它能够独立稳定存在，或者能够作为一个整体相对于整个蛋白质发生运动。

We have already encountered a well-studied motif, the coiled coil of α-keratin, which is also found in some other proteins. The distinctive arrangement of eight α helices in myoglobin is replicated in all globins and is called the globin fold. Note that a motif is not a hierarchical structural element falling between secondary and tertiary structure. It is simply a folding pattern.

我们前面已经遇到过一个研究得很透彻的模体——α-角蛋白的卷曲螺旋，它也出现在另外一些蛋白质中。肌红蛋白中八段 α 螺旋的独特排布方式在所有珠蛋白里重复出现，被称为珠蛋白折叠。要注意：模体并不是介于二级结构与三级结构之间的层级结构单元，它只是一种折叠模式。

In a Ramachandran plot, the dihedral angles that define the α helix and the β conformation fall within a relatively restricted range of sterically allowed structures (Fig. 4-8a). Most values of ϕ and ψ taken from known protein structures fall into the expected regions, with high concentrations near the α helix and β conformation values, as predicted (Fig. 4-8b). The only amino acid residue often found in a conformation outside these regions is glycine. Because its side chain is small, a Gly residue can take part in many conformations that are sterically forbidden for other amino acids.

在拉氏图中，规定 α 螺旋和 β 构象的那些二面角，落在空间位阻允许的一个相对狭窄的范围内（图 4-8a）。取自已知蛋白质结构的绝大多数 φ、ψ 数值都落在预期区域内，并如预测的那样在 α 螺旋和 β 构象对应数值附近高度集中（图 4-8b）。唯一经常出现在这些区域之外的氨基酸残基是甘氨酸。甘氨酸侧链很小，因此能采取许多对其他氨基酸而言因空间位阻而被禁止的构象。

The Ramachandran plot is a visual description of the combinations of ϕ and ψ dihedral angles that are permitted in a peptide backbone and those that are not permitted due to steric constraints. Dihedral angles that define the α helix and the β conformation are found only within certain regions of the plot.

拉氏图直观地描述了肽链主链上允许存在的 φ、ψ 二面角组合，以及因空间位阻而不允许存在的组合。规定 α 螺旋和 β 构象的那些二面角，只出现在图中特定的区域内。

Prion protein is a normal constituent of brain tissue in all mammals. Its role is not known in detail, but it may have a molecular signaling function. Strains of mice lacking the gene for PrP (and thus the protein itself) suffer no obvious ill effects. Illness occurs only when the normal cellular PrP, or PrPC, occurs in an altered conformation called PrPSc (Sc denotes scrapie). The structure of PrPC has two α helices. The structure of PrPSc is very different, with much of the structure converted to amyloidlike β sheets (Fig. 2).

朊病毒蛋白是所有哺乳动物脑组织的正常成分。它的作用尚不清楚，但可能有分子信号功能。缺失 PrP 基因（因而也没有该蛋白）的小鼠品系并无明显不良反应。只有当正常的细胞型 PrP（即 PrPC）呈现一种称为 PrPSc 的改变了的构象时，疾病才发生（Sc 指羊瘙痒病 scrapie）。PrPC 的结构含两段 α 螺旋；PrPSc 的结构则大不相同，其中大部分转变成类淀粉样的 β 折叠片（图 2）。

A misfolded brain protein seems to be the causative agent of several rare degenerative brain diseases in mammals. Perhaps the best known of these is bovine spongiform encephalopathy (BSE; also known as mad cow disease). Related diseases include kuru and Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. These diseases are also referred to as spongiform encephalopathies, because the diseased brain frequently becomes riddled with holes (Fig. 1).

一种错误折叠的脑蛋白，似乎是哺乳动物几种罕见脑退行性疾病的病因。其中最有名的是牛海绵状脑病（BSE，即疯牛病）。相关疾病还包括人的库鲁病和克-雅病、羊的瘙痒病，以及鹿和麋鹿的慢性消耗病。这些疾病也称为海绵状脑病，因为患病的脑常常布满空洞（图 1）。

It is a helical structure, and Pauling and Corey called it the α helix (Fig. 4-3). In this structure, the polypeptide backbone is tightly wound around an imaginary axis drawn longitudinally through the middle of the helix, and the R groups of the amino acid residues protrude outward from the helical backbone (Fig. 4-3b, c). The repeating unit is a single turn of the helix, which extends about 5.4 Å along the long axis, slightly greater than the periodicity that Astbury observed on x-ray analysis of hair keratin.

这是一种螺旋结构，鲍林和科里称之为 α 螺旋（图 4-3）。在该结构中，多肽主链紧紧缠绕在一条纵向贯穿螺旋中央的假想轴上，氨基酸残基的 R 基则从螺旋主链向外伸出（图 4-3b、c）。重复单位是螺旋的一圈，沿长轴延伸约 5.4 Å，略大于阿斯特伯里用 X 射线分析毛发角蛋白时观察到的周期。

Not all proteins fold spontaneously as they are synthesized in the cell. Folding for many proteins requires chaperones, proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironments in which folding can occur. Several types of molecular chaperones are found in organisms ranging from bacteria to humans. Two major families of chaperones, both well studied, are the Hsp70 family and the chaperonins.

并非所有蛋白质在细胞内合成时都能自发折叠。许多蛋白质的折叠需要分子伴侣——这类蛋白质与部分折叠或错误折叠的多肽相互作用，促成正确的折叠途径，或提供可供折叠进行的微环境。从细菌到人的各类生物中都发现了几种类型的分子伴侣。研究得比较透彻的两大家族是 Hsp70 家族和伴侣蛋白（chaperonins）。

Protein structures have evolved to function in particular cellular or extracellular environments. Conditions different from these environments can result in protein structural changes, large and small. A loss of three-dimensional structure sufficient to cause loss of function is called denaturation. The denatured state does not necessarily equate with complete unfolding of the protein and randomization of conformation. Under most conditions, denatured proteins exist in a set of partially folded states.

蛋白质的结构在进化中形成，以便在特定的细胞内或细胞外环境中行使功能。条件一旦偏离这些环境，就会引起蛋白质结构或大或小的改变。三维结构的丧失达到足以引起功能丧失的程度，就称为变性。变性状态并不一定等同于蛋白质完全去折叠、构象完全随机化。在大多数条件下，变性的蛋白质以一组部分折叠状态存在。

The Hsp70 proteins bind to and release polypeptides in a cycle that uses energy from ATP hydrolysis and involves several other proteins (including a class called Hsp40). Figure 4-28 illustrates chaperone-assisted folding as elucidated for the eukaryotic Hsp70 and Hsp40 chaperones. The binding of an unfolded polypeptide by an Hsp70 chaperone may break up a protein aggregate or prevent the formation of a new one. When the bound polypeptide is released, it has a chance to resume folding to its native structure. If folding does not occur rapidly enough, the polypeptide may be bound again and the process repeated. Alternatively, the Hsp70-bound polypeptide may be delivered to a chaperonin.

Hsp70 蛋白结合并释放多肽的循环利用 ATP 水解提供的能量，还需要另外几种蛋白质参与（包括一类称为 Hsp40 的蛋白）。图 4-28 用真核生物的 Hsp70 和 Hsp40 说明了分子伴侣辅助折叠的过程。Hsp70 分子伴侣结合未折叠的多肽，可以拆散已有的蛋白质聚集体，也可以防止新聚集体形成。被结合的多肽一旦释放，就有机会重新折叠成天然结构。若折叠进行得不够快，该多肽可能再次被结合，如此反复。或者，与 Hsp70 结合的多肽被递交给伴侣蛋白。

The term secondary structure refers to any chosen segment of a polypeptide chain and describes the local spatial arrangement of its main-chain atoms, without regard to the positioning of its side chains or its relationship to other segments. A regular secondary structure occurs when each dihedral angle, ϕ and ψ, remains the same or nearly the same throughout the segment.

二级结构一词是针对多肽链中任选的一段而言的，它描述该段主链原子的局部空间排布，既不考虑侧链的位置，也不考虑该段与其他肽段的关系。当一段肽链中每一个二面角 φ 和 ψ 都保持相同或几乎相同时，就出现规则二级结构。

In the α helix, the repeating unit is a single helical turn of ∼5.4 Å or 3.6 amino acids. The common form found in proteins is right-handed with the amino acid R groups protruding away from the helical backbone. The propensity of a protein segment to form an α helix depends on the composition of its amino acids and amino acid positions relative to one another and relative to the helical dipole. In the β conformation, amino acids are extended in a zigzag fashion. When several β strands are arranged adjacent to one another, they can form either parallel or antiparallel β sheets.

在 α 螺旋中，重复单位是一圈螺旋，约 5.4 Å 或 3.6 个氨基酸。蛋白质中常见的形式是右手螺旋，氨基酸的 R 基向外伸出、背离螺旋主链。一段肽链形成 α 螺旋的倾向，取决于其氨基酸的组成，以及各氨基酸相对彼此、相对螺旋偶极的位置。在 β 构象中，氨基酸以锯齿形伸展。若干条 β 股彼此相邻排列时，可以形成平行或反平行的 β 折叠片。

Many proteins have multiple polypeptide subunits (from two to hundreds). The association of polypeptide chains can serve a variety of functions. Many multisubunit proteins have regulatory roles; the binding of small molecules may affect the interaction between subunits, causing large changes in the protein’s activity in response to small changes in the concentration of substrate or regulatory molecules (Chapter 6). In other cases, separate subunits take on separate but related functions, such as catalysis and regulation. Some associations, such as those seen in the fibrous proteins considered earlier in this chapter and the coat proteins of viruses, serve primarily structural roles.

许多蛋白质含有多个多肽亚基（从两个到几百个）。多肽链的缔合可以实现多种功能。许多多亚基蛋白质具有调节作用：小分子的结合可以影响亚基之间的相互作用，使蛋白质的活性随底物或调节分子浓度的微小变化而发生很大改变（第 6 章）。另一些情况下，各亚基分别承担不同但相关的功能，例如催化与调节。还有一些缔合主要起结构作用，如本章前面讲过的纤维状蛋白质和病毒的外壳蛋白。

The hydrophobic effect, derived from the increase in entropy of the surrounding water when nonpolar molecules or groups are clustered together, makes the major contribution to stabilizing the globular form of most soluble proteins. Hydrogen bonds and ionic interactions are optimized in the thermodynamically most stable structures. Van der Waals interactions involve attractive forces between molecular dipoles that occur over short distances. Individually these interactions are weak, but they combine in well-packed protein structures to provide significant effects and stabilization.

疏水效应来自非极性分子或基团聚集时周围水的熵增，它对大多数可溶性蛋白质球状形态的稳定作出主要贡献。在热力学上最稳定的结构中，氢键和离子相互作用被优化到最佳状态。范德华相互作用是分子偶极之间在短距离内产生的吸引力。单个来看这些相互作用都很弱，但在堆积良好的蛋白质结构中它们叠加起来，就产生显著的效应和稳定作用。

One exciting application of computational biochemistry is the creation of “designer proteins.” These are proteins with completely novel folds or functions not yet identified in nature. Designer proteins have potential applications in bioengineering, medicine, materials science, and chemistry.

计算生物化学一个激动人心的应用，是创造「设计蛋白」。这类蛋白质具有自然界中尚未发现的全新折叠或全新功能。设计蛋白在生物工程、医学、材料科学和化学上都有潜在应用。

Foldit is a video game in which users compete to solve protein-folding puzzles (Fig. 1). Users are rewarded for making stabilizing contacts in the structure, like hydrogen bonds or favorable van der Waals interactions. Virtual protein designs (sometimes called theozymes) from the video game can then be tested in the real world. DNA genes coding for the virtual proteins can be created in the laboratory and be used to recombinantly produce the proteins in bacteria, using techniques described in Chapter 9. The purified proteins can then be structurally characterized by x-ray crystallography or NMR to see how closely the real-world structures and computationally designed structures match.

Foldit 是一款电子游戏，玩家在其中比赛解决蛋白质折叠难题（图 1）。玩家在结构中做出有稳定作用的接触，例如氢键或有利的范德华相互作用，就能得分。游戏里产生的虚拟蛋白质设计（有时称为 theozyme）随后可以在现实中检验：编码这些虚拟蛋白的 DNA 基因可在实验室构建，并用第 9 章介绍的技术在细菌中重组表达出蛋白质。纯化后的蛋白质再用 X 射线晶体学或 NMR 做结构表征，看现实中的结构与计算设计的结构吻合到什么程度。

The three-dimensional structure and the function of most proteins can be destroyed by denaturation, demonstrating a relationship between structure and function. Heat, extremes of pH, organic solvents, solutes, and detergents can all be used to denature proteins. Some denatured proteins can renature spontaneously to form biologically active protein, showing that tertiary structure is determined by amino acid sequence.

大多数蛋白质的三维结构和功能都可以被变性破坏，这说明结构与功能之间存在联系。加热、极端 pH、有机溶剂、某些溶质和去污剂都能使蛋白质变性。有些变性蛋白质能自发复性、重新形成有生物活性的蛋白质，这表明三级结构由氨基酸序列决定。

The tertiary structure of a globular protein is determined by its amino acid sequence. The most important proof of this came from experiments showing that denaturation of some proteins is reversible. Certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if they are returned to conditions in which the native conformation is stable. This process is called renaturation.

球状蛋白质的三级结构由它的氨基酸序列决定。这一点最重要的证据来自实验：某些蛋白质的变性是可逆的。某些球状蛋白质被加热、极端 pH 或变性试剂变性后，只要重新置于天然构象稳定的条件下，就能恢复其天然结构和生物活性。这一过程称为复性。

The physical environment in a crystal, of course, is not identical to that in solution or in a living cell. A crystal imposes a space and time average on the structure deduced from its analysis, and x-ray diffraction studies provide little information about molecular motion within the protein. The conformation of proteins in a crystal can also be affected by nonphysiological factors such as incidental protein-protein contacts within the crystal.

当然，晶体中的物理环境与溶液中或活细胞中的并不相同。晶体使人们由分析推出的结构在空间和时间上都被平均化，因此 X 射线衍射研究几乎给不出蛋白质内部分子运动的信息。晶体中蛋白质的构象还可能受到非生理因素的影响，例如晶体内偶然形成的蛋白质—蛋白质接触。

The unusual amino acid content of collagen is related to structural constraints unique to the collagen helix. The amino acid sequence in collagen is generally a repeating tripeptide unit, Gly–X–Y, where X is often Pro and Y is often 4-Hyp. Only Gly residues can be accommodated at the very tight junctions between the individual α chains (Fig. 4-11b). The Pro and 4-Hyp residues permit the sharp twisting of the collagen helix. The amino acid sequence and the supertwisted quaternary structure of collagen allow a very close packing of its three polypeptides.

胶原不同寻常的氨基酸组成，与胶原螺旋特有的结构限制有关。胶原的氨基酸序列一般是重复的三肽单位 Gly–X–Y，其中 X 常为 Pro，Y 常为 4-Hyp。三条 α 链彼此接触的连接处极为紧密，只能容纳 Gly 残基（图 4-11b）。Pro 和 4-Hyp 残基则使胶原螺旋能够急剧扭转。胶原的氨基酸序列和超螺旋的四级结构，使它的三条多肽链能极紧密地堆积在一起。

A region with more than 20 residues of high hydropathy index is presumed to be a transmembrane segment. When the sequences of membrane proteins of known three-dimensional structure are scanned using simple online bioinformatics tools, we find a reasonably good correspondence between predicted and known membrane-spanning segments. Hydropathy analysis predicts a single hydrophobic helix for glycophorin (Fig. 11-13a) and seven transmembrane segments for bacteriorhodopsin (Fig. 11-13b) — in agreement with structures known from x-ray crystallography.

一段含 20 个以上高疏水指数残基的区域，可推定为跨膜片段。用简单的在线生物信息学工具扫描已知三维结构膜蛋白的序列，可以发现预测的跨膜片段与已知跨膜片段吻合得相当好。疏水性分析预测血型糖蛋白只有一段疏水螺旋（图 11-13a），而细菌视紫红质有 7 段跨膜片段（图 11-13b）——这与 X 射线晶体学解出的结构一致。

Computational tools are now indispensable to biochemistry. While advances in computers and software have revolutionized how protein structures can be solved by x-ray crystallography, NMR, and cryo-EM, advances in computational chemistry have now allowed a number of studies of protein structure to be carried out entirely in silico using powerful molecular modeling and dynamics software.

计算工具如今已是生物化学不可或缺的手段。计算机与软件的进步彻底改变了用 X 射线晶体学、NMR 和冷冻电镜解析蛋白质结构的方式；与此同时，计算化学的进步已使一批蛋白质结构研究能够借助强大的分子建模与动力学软件，完全在计算机中（in silico）完成。

The stability of a protein is not simply the sum of the free energies of formation of the many weak interactions within it. For every hydrogen bond formed in a protein during folding, a hydrogen bond (of similar strength) between the same group and water was broken. The net stability contributed by a given hydrogen bond, or the difference in free energies of the folded and unfolded states, may be close to zero. Ionic interactions may be either stabilizing or destabilizing.

蛋白质的稳定性并不是其内部众多弱相互作用形成自由能的简单加和。折叠时每形成一个蛋白质内部的氢键，同一基团与水之间原有的一个强度相近的氢键就被破坏。因此某一个氢键所贡献的净稳定性，也就是折叠态与去折叠态的自由能之差，可能接近于零。离子相互作用则既可能起稳定作用，也可能起去稳定作用。

Globular proteins have more complicated tertiary structures, often containing several types of secondary structure in the same polypeptide chain, and fulfill many different functional roles in the cell. The first globular protein structure to be determined, by x-ray diffraction methods, was that of the O2-binding protein myoglobin. The myoglobin structure revealed for the first time how protein structure and function are connected.

球状蛋白质具有较复杂的三级结构，同一条多肽链中往往含有几种类型的二级结构，并在细胞中承担多种不同的功能角色。第一个用 X 射线衍射方法测定出结构的球状蛋白质，是结合 O2 的肌红蛋白。肌红蛋白的结构第一次揭示了蛋白质结构与功能是如何联系在一起的。

A classic example is the denaturation and renaturation of ribonuclease A, demonstrated by Christian Anfinsen in the 1950s. Purified ribonuclease A denatures completely in a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys residues, and the urea disrupts the stabilizing hydrophobic effect, thus freeing the entire polypeptide from its folded conformation. Denaturation of ribonuclease is accompanied by a complete loss of catalytic activity. When the urea and the reducing agent are removed, the denatured ribonuclease spontaneously refolds into its correct tertiary structure, with full restoration of its catalytic activity (Fig. 4-25).

一个经典例子是核糖核酸酶 A 的变性与复性，由 Christian Anfinsen 在 20 世纪 50 年代完成。纯化的核糖核酸酶 A 在高浓度脲溶液中、并有还原剂存在时完全变性。还原剂切断四对二硫键，生成八个 Cys 残基；脲则破坏起稳定作用的疏水效应，从而使整条多肽链摆脱其折叠构象。核糖核酸酶变性的同时，催化活性完全丧失。当脲和还原剂被除去后，变性的核糖核酸酶自发重新折叠成正确的三级结构，催化活性完全恢复（图 4-25）。