分子生物学Chapter 2 Protein Structure

Chapter 2 Protein Structure

Introduction

Sizes and shapes of proteins

Globlar proteins, including most enzymes, behave in solution like compact , roughly spherical particles. Fibrous proteins have a high axial ratio and are often of structural importance, for example fibroin and keratin. Sizes range from a few thousand to several million Daltons.

Some proteins contain bound nonprotein materials (prosthetic groups or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexs.

Shapes of proteins

Globular proteins: enzymes

Fibrous proteins: important structural proteins (silk fibroin, keratin in hair and wools )

Chapter 2 Protein Structure

1. Aminp Acids

R-CH(NH2)-COOH

a-carbon is chiral (asymmetric) except in glycine (R is H)

Amino acids can exit in both D- and L- stereoisomers, but only L-isomers are found in proteins

Amino acids are dipolar ions (zwitterions) in aqueous solution and are amphoteric .

The side chains (R) differ in size, shape, charge and chemical reactivity

A few proteins contain nonstandard amino acids that are formed by post-translational modification of the parent amino acids.

1.1Amino acids with charged side chains

“Basic” amino acids: containing positively charged groups

Lysine (Lys, K): a second amino group attached to the e-carbon atom

Arginine (arg, R): a guanidino group attached to the d-carbon atom

Histidine (His, H): a imidazole group has a p Ka near neutrality. This group can be reversibly protonated under physiological conditions, which contribute to the catalytic mechanism of many enzymes.

1.2. Amino acids with polar uncharged side chains (hydrophilic)--containing groups that form hydrogen bonds with water

Serine (Ser, S) & threonine (Thr, T) have hydroxyl groups.

1.3. Amino acids with nonpolar aliphatic side chains (hydrophobic )

Alkyl side chains

1.4. Amino acids with aromatic side chains (hydrophobic )

2. Primary structure

Amino acids are linked by peptide bonds between alpha-carboxyl and alpha-amino groups. The resulting polypeptide sequence has an N terminus and a C terminus.

Polypeptides commonly have between 100 and 1500 amino acids linked in this way

Formation of a peptide bond (shaded in gray) in a dipeptide.

The amino acid in a peptide is also called a residue.

The smallest amino acid, glycine, has a single hydrogen atom as its R group. Its small size allows it to fit into tight spaces. Unlike any of the other common amino acids, proline has a cyclic ring that is produced by formation of a covalent bond between its R group and the amino group on Cα. Proline is very rigid, and its p resence creates a fixed kink in a protein chain. Proline and glycine are sometimes found at points on a protein's surface where the chain loops back into the protein

3. Secondary Structure

Secondary structure refers to the localized organization of parts of a polypeptide chain, which can assume several different spatial arrangements. A single polypeptide may exhibit all types of secondary structure. Without any stabilizing interactions, a polypeptide assumes a random-coil structure.

3.1 Model of the a helix.

The polypeptide backbone is folded into a spiral that is held in place by hydrogen bonds (black dots) between backbone oxygen atoms and hydrogen atoms. All the hydrogen bonds have the same polarity. The outer surface of the helix is covered by the side-chain R groups.

a-helix

every 3.6 residues make one turn,

the distance between two turns is 0.54 nm,

the C=O (or N-H) of one turn is hydrogen bonded to N-H (or C=O) of the

neighboring

3.2 Beta Strand and Beta Sheet

In a β strand, the torsion angle of N-Cβ-C-N in the backbone is about 120 degrees. The following figure shows the conformation of an ideal β strand.Note that the sidechains of two neighboring residues project in the opposite direction from the backbone.

Beta sheet

A β sheet consists of two or more hydrogen bonded β strands.The two neighboring β strands may be parallel if they are aligned in the same direction from one terminus (N or C) to the other, or anti-parallel if they are aligned in the opposite direction.

b SHEETS. (a) A simple two-stranded b sheet with antiparallel b strands. A sheet is stabilized by hydrogen bonds (black dots) between the b strands. The planarity of the peptide bond forces a b sheet to be pleated; hence, this structure is also called a b pleated sheet, or simply a pleated sheet. (b) Side view of a b sheet showing how the R groups protrude above and below the plane of the sheet. (c) Model of binding site in class I MHC (major histocompatibility complex) molecules, which are involved in graft rejection. A sheet comprising eight antiparallel b strands (green) forms the bottom of the binding cleft, which is lined by a pair of a helices (blue). A disulfide bond is shown as two connected yellow spheres. The MHC binding cleft is large enough to bind a peptide 8 10 residues long.

3.3 Turns

Composed of three or four residues, turns are compact, U-shaped secondary structures stabilized by a hydrogen bond between their end residues. They are located on the surface of a protein, forming a sharp bend that redirects the polypeptide backbone back toward the interior. Glycine and proline are commonly present in turns.

4. Tertiary Structure

Tertiary structure, the next-higher level of structure, refers to the overall conformation of a polypeptide chain, that is, the three-dimensional arrangement of all the amino acids residues. In contrast to secondary structure, which is stabilized by hydrogen bonds, tertiary structure is stabilized by hydrophobic interactions between the nonpolar side chains and, in some proteins, by disulfide bonds. These stabilizing forces hold t he α helices, β strands, turns, and random coils in a compact internal scaffold

The ribbon representation of the 3D structure of RNase A.

Noncovalent interaction between side chains that hold the tertiary structure together:van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions

Covalent interaction: disulfide bonds

Denaturation of protein by disruption of its 2o and 3o structure will lead to a random coil conformation

5. Quaternary structure

Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary

structure.

Prosthetic groups

Prosthetic groups covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions.

Examples : heme groups in hemogobin

O rganization of the catabolite activator

protein (CAP)

6. Domains, motifs and families

6.1 Protein Motifs

A motif is a characteristic domain structure consisting of two or more α helices or β strands.

Common examples include coiled coil, helix-turn-helix helix-loop-helix, zinc finger, leucine zipper etc.

Many proteins contain one or more motifs built from particular combinations of secondary structures.

A motif is defined by a specific combination of secondary structures that has a particular topology and is organized into a characteristic three-dimensional structure. Structural motifs:

?Groupings of secondary structural elements that frequently occur in globular proteins

?Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family ?Represent the best solution to a structural-functional requirement

βαβ motif

6.2 Domains:

Structurally independent units of many proteins, connected by sections with limited higher order structure within the same polypeptide.

They can also have specific function such as substrate binding

Functional Domains

Domains sometimes are defined in functional terms based on observations that the activity of a protein is localized to a small region along its length. For instance, a particular region or regions of a protein may be responsible for its catalytic activity (e.g., a kinase domain) or binding ability (e.g., a DNA-binding domain, membrane-binding domain).

Modules:

The organization of tertiary structure into domains further illustrates the principle that complex molecules are built from simpler components. Like secondary-structure motifs, tertiary-structure domains are incorporated as modules into different proteins, thereby modifying their functional activities. The modular approach to protein architecture is particularly easy to recognize in large proteins, which tend to be a mosaic of different domains and thus can perform different functions simultaneously Schematic diagrams of various proteins, illustrating their modular nature.

Epidermal growth factor (EGF) is generated by proteolytic cleavage of a precursor protein containing multiple EGF domains (orange). The EGF domain also occurs in Neu protein and in tissue plasminogen activator (TPA). Other domains, or modules, in these proteins include a chymotryptic domain (purple), an immunoglobulin domain (green), a fibronectin domain (yellow), a membrane-spanning domain (pink), and a kringle domain (blue).

6.3 Protein families:

Structurally and functionally related proteins from different sources

Evolutionary tree showing how the globin protein family arose, starting from the most primitive oxygen-binding proteins, leghemoglobins, in plants.Sequence comparisons have revealed that evolution of the globin proteins parallels the evolution of vertebrates. Major junctions occurred with the divergence of myoglobin from hemoglobin and the later divergence of hemoglobin into the a and b subunits.

Domains, motifs and families

Domains form semi-independent structural and functional units within a single polypeptide chain. Domains are often encoded by individual exons within a gene. New proteins may have evolved through new combinations of exons. Motifs are groupings of secondary structural elements or amino acid sequences often found in related members of protein families. Similar structural motifs are also found in proteins which have no sequence similarity. Protein families arise through gene duplication and subsequent divergent evolution of the new genes.

7. Folding of Proteins

The amino acid sequence of a protein determines its folding.

Folding of proteins in vivo is promoted by chaperones.

Protein Folding:

chaperones are involved in vivo

(1)The rapidly reversible formation of local secondary structure

(2)Formation of domains through the cooperative aggregation of folding

nulcei

(3)Assembly of domains into a “molten” globule

(4)Conformational adjustment of the monomer

(5)Final conformational adjustment of the dimeric protein to form the native

structure.

There are two general families of chaperones:

Molecular chaperones,which bind and stabilize unfolded or partially folded proteins, thereby preventing these proteins from being degraded;

Chaperones have ATPase activity, and their ability to bind and stabilize their target proteins is specific and dependent on ATP hydrolysis.

Chaperonins, which directly facilitate their folding.

Molecular chaperones consist of the Hsp70 family of proteins, which includes Hsp70 in the cytosol and mitochondrial matrix, Bip in the endoplasmic reticulum, and DnaK, a bacterial chaperone. When bound to ATP, Hsp70 assumes an open form in which an exposed hydrophobic pocket transiently binds to exposed

hydrophobic regions of the unfolded target protein. Hydrolysis of the bound ATP causes Hsp70 to assume a closed form, releasing the target protein.

Proper folding of a small proportion of proteins (e.g., the cytoskeletal proteins actin and tubulin) requires additional assistance, which is provided by chaperonins.

Eukaryotic chaperonins, called TCiP, are large, barrel-shaped, multimeric complexes composed of eight Hsp60 units. The bacterial homolog, known as GroEL, contains 14 identical subunits.

The GroEL folding mechanism: In bacteria, a partially folded or misfolded polypeptide is inserted into the cavity of GroEL, where it binds to the inner wall and folds into its native conformation. In an ATP-dependent step, GroEL expands and the protein exits GroEL, a process assisted by a co-chaperonin, GroES, which caps the ends of GroEL . Because the eukaryotic chaperonin TCiP lacks a GroES-type co-chaperonin, the last step must differ in eukaryotes. Moreover, the size of the cavity in TCiP limits this folding pathway to polypeptides smaller than 55 kDa.

8. Chemical Modifications and Processing

Chemical modification involves the linkage of a chemical group to the terminal amino or carboxyl groups or to reactive groups in the side chains of internal residues; in some cases, these modifications are reversible.

Acetylation,the addition of an acetyl group (CH3CO) to the amino group of the N-terminal residue is the most common form of chemical modification, involving an estimated 80 percent of all proteins

The internal residues in proteins can be modified by attachment of a variety of chemical groups to their side chains.

The most important modification is phosphorylation of serine, threonine, and tyrosine residues. There are numerous examples of proteins whose activity is regulated by reversible phosphorylation and dephosphorylation.

The side chains of asparagine, serine, and threonine are sites for glycosylation, the attachment of linear and branched carbohydrate chains. Many secreted proteins and membrane proteins contain glycosylated residues.

Processing:

In the most common form of processing, residues are removed from the C- or N-terminus of a polypeptide by cleavage of the peptide bond in a reaction catalyzed by proteases. Proteolytic cleavage is a common mechanism of activation or inactivation, especially of enzymes involved in blood coagulation or digestion.

An unusual type of processing, termed protein selfsplicing, occurs in bacteria and primitive eukaryotes. Splicing refers to a process analogous to editing film: an internal segment of polypeptide, an intein,is removed and the ends of the polypeptide are rejoined. Unlike proteolytic processing,

9. Degrading Proteins

Cells have both extracellular and intracellular pathways for degrading proteins Extracellular pathways for degrading proteins

The major extracellular pathway is the system of digestive proteases, which break down ingested proteins to polypeptides in the intestinal tract. These include endoproteases such as trypsin and chymotrypsin, which cleave the protein backbone adjacent to basic and aromatic residues; exopeptidases,which sequentially remove residues from the N-terminus (aminopeptidases) or C-terminus (carboxypeptidases) of proteins; and peptidases,which split oligopeptides into di- and tripeptides and

individual amino acids.

Intracellular pathway:

One major intracellular pathway involves degradation by enzymes within lysosomes, membrane-limited organelles whose interior is acidic . Distinct from the lysosomal pathway are cytosolic mechanisms for degrading proteins.

The ubiquitin-mediated pathway

addition of a chain of ubiquitin molecules to an internal lysine side chain of a target protein and proteolysis of the ubiquitinated protein by a proteasome, a large, cylindrical multisubunit complex

A conjugating enzyme catalyzes formation of a peptide bond between ubiquitin (Ub) and the side-chain –NH2 of a lysine residue in a target protein. Additional Ub molecules are added, forming a multiubiquitin chain. This chain is thought to direct the tagged protein to a proteasome, which cleaves the protein into numerous small peptide fragments.

To be targeted for degradation by the ubiquitin-mediated pathway, a protein must contain a structure that is recognized by a ubiquitinating enzyme complex. Different conjugating enzymes recognize different degradation signals in target proteins.

For example, the internal sequence Arg-X-X-Leu-Gly-X-Ile-Gly-Asx in mitotic cyclin is recognized by the ubiquitin-conjugating enzyme E1.

Internal sequences enriched in proline, glutamic acid, serine, and threonine (PEST sequences) are recognized by other enzymes.