1. Metals in proteins - which and why?
Life requires an interplay between organic (e.g., proteins) and inorganic (e.g., metals) matter. At present, we are aware of at least thirteen metals which are essential for plants and animals (Bertini et al. 1994; Lippard and Berg 1994). Four of these, sodium, potassium, magnesium and calcium, are present in large quantities and are known as bulk metals (Fenton 1995). The remaining nine, which are present in small quantities, are the d-block elements vanadium, chromium, molbydenum, manganese, iron, cobalt, nickel, copper and zinc, and are known as the trace metals. The bulk metals form 1-2 % of the human body-weight whereas the trace elements represent less than 0.01 %. Even of iron, the most widely used trace metal, we need only of the order of 4-5 grams in a human body (Fenton 1995). The concentrations of metals in the cells are strictly regulated at their respective optimum levels: too much or too little is often harmful and may even be lethal to the organism. Most of the trace metals are found as natural constituents of proteins. In this way, Nature has taken advantage of the special properties of the metal ions and tuned them by protein encapsulation to perform a wide variety of specific functions associated with life processes (Bertini et al. 1994; Lippard and Berg 1994).
More than 30 % of all proteins in the cells exploit one or more metals to perform their specific functions (Gray 2003); over 40 % of all enzymes contain metals (Bertini et al. 1994; Lippard and Berg 1994). The amino-acids that regularly act as metal ligands in proteins are thiolates of cysteines, imidazoles of histidines, carboxylates of glutamic and aspartic acids, and phenolates of tyrosines. Each metal is considered a Lewis acid and favors different sets of protein ligands; the preferences are frequently dictated by the hard-soft theory of acids and bases. The coordination number and geometry of each metal site is determined by the metal's oxidation state albeit substantial distortions from the idealized structures can and do occur in metalloproteins (Bertini et al. 1994; Lippard and Berg 1994). Due to unique chemical properties of each metal, different metals are apt for different types of biological functions, although there is overlap is most cases.
Iron and copper are redox-active metals and often participate in electron transfer (Fenton 1995). In respiration and photosynthesis processes, small redox-active metalloproteins facilitate electron-transfer reactions by alternately binding to specific integral membrane proteins that often contain several metal sites. Well-known examples of soluble redox-active metalloproteins are iron-sulfur-cluster proteins, heme-binding cytochromes and blue-copper proteins. Iron and copper are also involved in dioxygen (O2) storage and carriage via metalloproteins (e.g., hemoglobin, myoglobin and hemocyanin). In contrast to iron and copper, zinc serves as a superacid center in several metalloenzymes, promoting hydrolysis or cleavage of a variety of chemical bonds. Representative proteins that use catalytic zinc ions are carboxypeptidases, carbonic anhydrase, and alcohol dehydrogenase. In addition, zinc ions often play structural roles in proteins (e.g. in superoxide dismutase and zinc-finger motifs). Most of the other trace metals have been identified as parts of metalloenzymes (Gray 2003; Lippard and Berg 1994). For example, manganese is found as a cofactor in mitochondrial superoxide dismutase, inorganic phosphatase and most notably photosynthesis system II. Nickel functions in enzymes such as urease and several hydrogenases. Both mobydenum and vanadium are found in nitrogenases, where they are present in larger clusters containing also iron and sulfur ions (Fenton 1995).
2. How are metals incorporated into appropriate proteins?
Despite this wealth of information of high-resolution structures of many metalloproteins, the folding-binding pathways for biosynthesis of metalloproteins are mostly unknown (Wittung-Stafshede 2002). Although unique features of different proteins result in selectivity for a particular metal, this selection is imperfect since proteins are dynamic molecules, something that is especially true for newly synthesized (unfolded) polypeptides. Protein affinity for metals tends to follow a universal order of preference, which for divalent metals is the Irving-Williams series. Thus, it remains a question how cells can contain proteins with weak-binding metals while simultaneously contain proteins requiring tight-binding metals? Intuitively, all metalloproteins would bind the most competitive metal. One solution to this paradox is the idea that the compartment (and its metal composition) in which the protein folds overrides the protein's metal-binding preference to control its metal content (Tottey et al. 2008). Another explanation is the fact that metal insertion into many proteins is strictly controlled by specific or unspecific protein-based delivery system.
In the case of copper, such ions are almost non-existent in their free form in the cytoplasm (O'Halloran and Culotta 2000) since copper's redox properties may result in oxidative damage of proteins, lipids, and nucleic acids. Instead, the cellular copper concentration is strictly controlled and most copper ions are delivered to their destinations by copper-chaperones (Lamb et al. 1999; Lamb et al. 2001). In the case of iron, transferrins transport iron into cells and releases it within the endosome; the protein hemopexin delivers heme to the same compartment. In the case of c-type cytochromes, it has been proposed that heme attachment is a required step before correct folding occurs in vivo (Bertini et al. 1994; Lippard and Berg 1994). The heme is covalently attached by a heme-lyase enzyme (Ramseier, Winteler, and Hennecke 1991) when the polypeptide is associated with the membrane. After heme insertion, the holo-protein is released from the membrane and is free to fold into its native configuration. While specific heme transporters and ligation enzymes appear necessary for c-type cytochrome assembly, a different, perhaps non-specific or diffusional, mechanism for heme transport and insertion into perplasmic heme proteins has been proposed based on E coli studies (Goldman, Gabbert, and Kranz 1996). The synthesis of iron-sulfur clusters in vivo requires a complex machinery encoded in prokaryotes by the isc (iron-sulfur-cluster) operon (Agar et al. 2000; Ollagnier-De Choudens et al. 2000; Ollagnier-de-Choudens et al. 2001). In eukaryotes, mitochondrial proteins, highly homologous to the bacterial ones have been shown to be involved in Fe-S cluster assembly. Although there are no known zinc-chaperones, membrane-transport and regulatory proteins specific for zinc have been identified in some organisms.
3. Roles of metals in protein folding and misfolding?
Since metal-binding proteins fold in a cellular environment where their cognate cofactors are present, either free in the cytoplasm or bound to delivery proteins, the question arises as to when metals bind to their corresponding proteins. Specifically, do they bind before, during or after polypeptide folding? As demonstrated in vitro, many metalloproteins retain strong metalloligand binding after polypeptide unfolding (Robinson et al. 1997; Bertini et al. 1997; Wittung-Stafshede et al. 1999; Wittung-Stafshede et al. 1998). This implies that in vivo metals may interact with their corresponding proteins before polypeptide folding takes place which could impact the folding reaction. Local and non-local structure in the unfolded protein may form due to specific coordination of the cofactor (Pozdnyakova, Guidry, and Wittung-Stafshede 2000). Such structural restriction of the ensemble of conformations may dramatically decrease the entropy of the unfolded state, limiting the conformational search for the native state (Luisi, Wu, and Raleigh 1999). The metal may this way serve as a nucleation site that directs polypeptide folding along a specific pathway in the free-energy landscape. In this regard, considering the in vivo biosynthesis, one must take into account that proteins are made on ribosomes and if metals may bind before, during or after release of the polypeptide from the ribosome. It has been proposed that polypeptides obtain structure as well as altered dynamics during the translational process (Ellis et al. 2008; Ellis, Culviner, and Cavagnero 2009). It is clear that metals in many cases stabilize final folded protein structures, but less is known about how they may modulate folding pathways, speed and folding-transition state ensembles.
Metals are often yin-yang elements: they play essential roles as cofactors in protein, but are often toxic in large amount and/or when free in biological fluids. A number of diseases (for example, Menke's syndrome and Wilson's disease) have been linked to alterations in cofactor-protein interactions (Rae et al. 1999). In addition, Cu2+ and Zn2+ ions have been shown to induce aggregation of amyloid-forming peptides (Villanueva et al. 2004). This underscores the fundamental importance of revealing the physical principles for metal interactions with folded, unfolded, and intermediate states of polypeptides.
4. Metals as modulators of protein conformations?
5. Organization and purpose of this book
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