Protein Structure, Stability, and Folding PDF

**4º Structure of Hemoglobin** Most proteins, particularly those with molecular masses \>100 kD, consist of more than one polypeptide chain/subunits (**oligomers**) that associate with specific geometry. The spatial arrangement of these subunits is known as a **protein's quaternary structure**. Sub...

**4º Structure of Hemoglobin** Most proteins, particularly those with molecular masses \>100 kD, consist of more than one polypeptide chain/subunits (**oligomers**) that associate with specific geometry. The spatial arrangement of these subunits is known as a **protein's quaternary structure**. Subunit construction of enzymes provide structural basis for regulation of their activities. Like multisubunit protein has subunit composition of α~2~β~2.~ **\ Symmetries of Oligomeric Proteins** Subunits are symmetrically arranged. Proteins can have only rotational symmetry. **Protein stability:** Proteins are stabilized by several forces. The **hydrophobic effect** (which causes nonpolar substances to minimize their contact with water is the major determinant of native protein structure) the **greatest influence on protein stability**. The combined hydrophobic and hydrophilic tendencies of individual amino acid residues in protein can be expressed as hydropathies. Look at the table for hydropathy scale of amino acid side chains. The greater a side chain's hydropathy, the more likely it is to occupy the interior of a protein and vise versa. **Hydropathy Plot:** **Bovine Chymotrypsinogen** Hydropathies are good predictors of which portions of a polypeptide chain are inside a protein, out of contact with aqueous solvent and which portions are outside. Figure here shows a hydropathic index plot for bovine chymotrypsinogen. The sum of the hydropathies of nine consecutive residues is plotted versus residue sequence number. A large positive hydropathic index indicates a hydrophobic region of the polypeptide, whereas a large negative value indicates a hydrophilic region. The upper bas denote the protein's interior regions. As determined by X-ray crystallography and the lower bars denote the protein's exterior region. **Ion Pairs in Myoglobin** Electrostatic interactions contribute to protein stability. The association of two ionic protein groups of opposite charge (e.g., Lys and Asp) is known as an ion pair or a salt bridge. About 75% of charged residues in protein are members of ions pairs that are located mostly on the protein surface. Examples of ion pair in myoglobin. In each case, oppositely charges\]d side chain groups from residue far apart in sequence closely approach each other through the formation of ion pairs. Despite the strong electrostatic attraction between the oppositely charged members of an ion pair, these interactions contribute little to the stability of a native protein. Disulfide bonds within and between polypeptide chains form as a protein folds into its native conformation. Important for locking in a particular backbone folding pattern as the protein proceeds from its fully extended state to its mature form. **Metal Ion Stabilized Zinc Finger** Metal ions stabilize some small domains. It may also function to internally cross-link proteins. Like at least 10 motifs collectively known as zinc fingers have been describes in nucleic acid binding proteins. These structures contain about 25 to 60 residues arranged around one or two Zn^2+^ ions that are tetrahedrally coordinated by the side chains of Cys, His, and occasionally Asp and Glu. **Protein denaturation** Proteins can be denatured by: 1. Heating. 2. pH variations. 3. Detergents. 4. Chaotropic agents (guanidium ion and urea) **Denaturation and Renaturation of RNase A** Protein can be renatured (denatured protein regains its native, functional three-dimensional structure after the removal of the denaturing agent). Christian Anfinsen experiment on Ribonuclease A (RNase A) showed that proteins ca be denatured reversibly. RNase A, a 124-residue single chain protein, is completely unfolded and its four disulphide bonds reductively cleaved in an 8M urea solution containing 2-mercaptoethanol. Dialyzing away the urea and reductant and exposing the resulting solution to O~2~ at pH 8 (which oxidizes the SH group to form disulfides) yields a protein that is 100% enzymatically active and physically indistinguishable from native RNase A. So Anfinsen's work demonstrated that protein can fold spontaneously into their native conformation and protein's primary structure indicates its 3D structure. **Molecular Dynamics of Myoglobin: Proteins "Breathing"** Calculations by Martin Karplus indicated that a protein's native structure probably consist of a large collection of rapidly interconverting conformations that have essentially equal stabilities. Conformational flexibility or **breathing** with structural displacement of up to \~2 A◦ allow small molecules to diffuse in and out of the interior of certain protein. Figure shows molecular dynamics of myoglobin. Several 'snapshots' of the protein calculated at intervals of 5x10^-12^ s are superimposed. The backbone is blue, the heme is yellow , and the His side chain linking the heme to the protein is orange. **Intrinsically Disordered Proteins: CREB** Intrinsically disordered proteins often adopt a specific secondary or tertiary structure when they bind to other molecules such as ions, organic molecules, proteins and nucleic acid. Example, transcription factor CREB (cyclic AMP response element-binding protein is disordered when free in solution but folds to an ordered conformation when it interacts with the CREB-binding protein. **Hypothetical Protein Folding Pathway** Protein follow folding pathways. Experiments have shown that many protein fold to their native conformations in less than a few seconds (within 5 ms). A hypothetical folding pathway is diagrammed in this figure. It shows a linear pathway for folding a two-domain protein. Folding begins with formation of secondary structures, it is extremely rapid. The driving force of folding is termed **hydrophobic** **collapse**. The collapsed state is known as **molten globule**. Over next 5 to 1000 ms, the secondary structure is stabilized, and tertiary structure begins to form, forming subdomains and domains. Slight conformational adjustment is done to produce native tertiary and quaternary structure. **Energy-Entropy Diagram: Protein Folding Funnel** A folding protein protein must proceed from a high-energy, high entropy state to low --energy, low entropy state. This energy-entropy relationship, which is diagramed here is known as **folding funnel.** The surface of the folding funnel represents all possible conformations that the polypeptide can assume with each point on it corresponding to a specific conformation. The height of each point is indicative of the conformation's energy, and the funnel width's at that energy is indicative of the polypeptide's entropy (no. of different conformations it can assume with that energy). The unfolded polypeptide proceeds from a high-energy, high-entropy(wide), disordered state to a low energy, low --entropy native conformation. Folding can occur via multiple trajectories, each starting from a different unfolded structure at the top of energy landscape. It has many high energy partially folded structures and few low energy native structures. **Protein Disulfide Isomerase Catalyzes Disulfide Interchange** **Protein disulfide isomerase (PDI) acts during protein folding**. PDI reacts with a disulfide group on the polypeptide to form a mixed disulfide and a Cys-SH group on the polypeptide. Another sulfide group on the polypeptide brought into proximity by the spontaneous folding of the polypeptide, it is attacked by Cys-SH group. The newly liberated Cys-SH group repeat this process with another disulfide bond ultimately yielding polypeptide containing only native disulfide bonds along with regenerated PDI. Oxidized (disulfide containing) PDI also catalyzes the initial formation of a polypeptide's disulfide bonds by a similar mechanism. Molecular chaperones are essential proteins that bind to unfolded and partially folded polypeptide chains to disrupt the improper association of exposed hydrophobic segments that would otherwise lead to non-native folding as well as polypeptide aggregation and precipitation. **Protein Folding & Disease** Many diseases are caused by protein misfolding. At least 35 different and usually fatal human diseases are associated with the extracellular deposition of normally soluble proteins in certain tissues in the form of insoluble fibrous aggregates. These aggregates are known as **amyloids**. The diseases are known as [amyloidosis], are set or rare inherited diseases in which mutant form of normally occurring proteins [lysozyme], [fibrinogen] accumulate in variety of tissues as amyloids. Alzheimer's disease is a neurodegenerative condition that strikes mainly the elderly causing mental deterioration and eventually death. It is characterized by brain tissue containing abundant amyloid plaques (deposits)surrounded by dead and dying neurons. Figure shows brain tissue from an individual with Alzheimer\'s disease. The two circular objects in this photomicrograph are plaques that consist of amyloid deposits of **Aβ** protein surrounded by halo neurites (axon and dendrites) from dead and dying neurons. **Amyloid-ϐ protein (Aβ)** protein are fibrils of 40 to 42 residue protein. Aβ is a fragment of Aβ precursor protein (AAP ), it is excised from AAP by actions of enzymes ϐ and γ secretases. Prion diseases are infectious. Caused by Prions (proteinaceous infectious particles). In this neuron develop large vacuoles that give brain tissue a spongelike microscopic appearance. The associated diseases are called as transmissible spongiform encephalopathies (TSEs), which include bovine spongiform enthcephalopathy, kuru, Creutzfeldt-Jakob-disease (CJK) caused in humans. And Scrapie in sheep and goat. The Scrapie prion (PrP) consist mostly hydrophobic residue. The hydrophobicity causes partially proteolyzed PrP to aggregate. Human PrP^C^ (normal cellular PrP) consists of a disordered 98 residue N terminal tail and 110 residue C terminal domain containing three α helices and a short two stranded antiparallel β sheet. Scrapie form pf PrP^Sc^ is identical to normal PrP in sequence but differs in secondary and tertiary structure. a\) The NMR structure of human prion protein (PrP^C^). The protein missing its first 23 residues, is drawn in ribbon form with helices red, ϐ sheet green, and other segments orange. Its disulfide bond is shown in yellow. B) a plausible model for the structure of PrP^Sc^. Note the formation of flexibly disordered N-terminal region. The high β sheet content of PrP^Sc^ presumably facilitates the aggregation of PrP^Sc^ as amyloid fibrils. **Amyloid Fibril Model** Spectroscopic analysis of amyloid fibrils indicates that they are rich in ϐ structure, withy individual β strands oriented perpendicular to the fiber axis. Figure shows model of an amyloid fibril. A) the model based on X-ray fiber diffraction measurements, is viewed normal to the fibril axis (above) and along the fibril axis (below). The arrow heads indicate the path but not necessarily the direction of the ϐ strands. B) A single β sheet, which is shown for clarity.