Biochemistry Chapter 4

What was Anfinsens plan to determine the conditions required to restore the tertiary structure.

Anfinsen's plan was to destroy the three-dimensional structure of the enzyme ( ribonuclease) and to then determine the conditions required to restore the tertiary structure. Chaotropic agents, such as urea, which disrupt all of the noncovalent bonds in a protein, were added to a solution of the ribonuclease. The disulfide bonds where then cleaved reversibly with a sulfhydryl reagent such as β-mercaptoethanol (Figure 4.32). In the presence of a large excess of β-mercaptoethanol, the disulfides (cystines) are fully converted into sulfhydryls (cysteines).

intrinsically unstructured proteins (IUPs).

As the name suggests, these proteins, completely or in part, do not have a discrete three-dimensional structure under physiological conditions. Indeed, an estimated 50% of eukaryotic proteins have at least one unstructured region greater than 30 amino acids in length. These proteins assume a defined structure on interaction with other proteins. This molecular versatility means that one protein can assume different structures and interact with different partners, yielding different biochemical functions. IUPs appear to be especially important in signaling and regulatory pathways.

Knowing amino acid sequences is important for several reasons.

First, amino acid sequences determine the three-dimensional structures of proteins. Second, knowledge of the sequence of a protein is usually essential to elucidating its mechanism of action (e.g., the catalytic mechanism of an enzyme). Third, sequence determination is a component of molecular pathology, a rapidly growing area of medicine. Fourth, the sequence of a protein reveals much about its evolutionary history. Proteins resemble one another in amino acid sequence only if they have a common ancestor. Consequently, molecular events in evolution can be traced from amino acid sequences; molecular paleontology is a flourishing area of research.

What are the rules governing the relation between an amino acid sequence and the three-dimensional structure of a protein?

First, the peptide bond is essentially planar (Figure 4.6). Thus, for a pair of amino acids linked by a peptide bond, six atoms lie in the same plane: the α-carbon atom and CO group of the first amino acid and the NH group and α-carbon atom of the second amino acid. Second, the peptide bond has considerable double-bond character owing to resonance structures: the electrons resonate between a pure single bond and a pure double bond.

Can a polypeptide chain fold into a regularly repeating structure?

In 1951, Linus Pauling and Robert Corey proposed that certain polypeptide chains have the ability to fold into two periodic structures called the α helix (alpha helix) and the β pleated sheet (beta pleated sheet). Subsequently, other structures such as turns and loops were identified

Two configurations are possible for a planar peptide bond which are...

In the trans configuration, the two α-carbonatoms are on opposite sides of the peptide bond. In the cis configuration, these groups are on the same side of the peptide bond. Almost all peptide bonds in proteins are trans.

Osteogenesis imperfecta

In this condition, which can vary from mild to very severe, other amino acids replace the internal glycine residue. This replacement leads to a delayed and improper folding of collagen, and the accumulation of defective collagen results in cell death. The most-serious symptom is severe bone fragility. Defective collagen in the eyes causes the whites of the eyes to have a blue tint (blue sclera).

How does a protein make the transition from an unfolded structure to a unique conformation in the native form?

Levinthal's paradox and Anfinsen's results suggest that proteins do not fold by trying every possible conformation; rather, they must follow at least a partly defined folding pathway consisting of intermediates between the fully denatured protein and its native structure.

The enormous difference between calculated and actual folding times is called ...

Levinthal's paradox.

phi rotation

Phi (ϕ) is the angle of rotation about the bond between the nitrogen and the α-carbon atoms.

Prions

Prions are composed largely or completely of a cellular protein called PrP, which is normally present in the brain. The prions are aggregated forms of the PrP protein termed PrPSC.

The ϕ and ψ values of possible conformations can be visualized on a two-dimensional plot called a

Ramachandran diagram

Ribonuclease

Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds (Figure 4.31).

The structure of a reverse turn

The CO group of residue i of the polypeptide chain is hydrogen bonded to the NH group of residue i + 3 to stabilize the turn

How is the elaborate three-dimensional structure of proteins attained?

The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation.

metamorphic protein

These proteins appear to exist in an ensemble of structures of approximately equal energy that are in equilibrium.

Each point on the surface ( funnel) represents a

a possible three-dimensional structure and its energy value. The funnel suggests that there are alternative pathways to the native structure.

Alpha helices, β pleated sheets, and turns are formed by...

a regular pattern of hydrogen bonds between the peptide NH and CO groups of amino acids that are often near one another in the linear sequence, or primary structure. Such regular folded segments are called secondary structure.

Myoglobin

a single polypeptide chain of 153 amino acids, is an oxygen-binding protein found predominantly in heart and skeletal muscle; it appears to facilitate the diffusion of oxygen from the blood to the mitochondria, the primary site of oxygen utilization in the cell.

The breadth of the funnel represents

all possible conformations of the unfolded protein.

Quaternary structure refers to the

arrangement of subunits and the nature of their interactions. The interactions among subunits of proteins displaying quaternary structure are usually the weak interactions discussed in Chapter 2: hydrogen bonds, ionic bonds, and van der Waals interactions.

Each residue contains a

carbonyl group... C=O, which is a good hydrogen acceptor and, with the exception of proline, an amino group (N — H) group, which is a good hydrogen-bond donor.

Stanley Prusiner found that..

certain infectious neurological diseases were found to be transmitted by agents that were similar in size to viruses but consisted only of protein. These diseases include bovine spongiform encephalopathy (commonly referred to as mad cow disease) and the analogous diseases in other organisms, including Creutzfeldt-Jakob disease (CJD) in human beings and scrapie in sheep. The agents causing these diseases are termed prions.

Myoglobin, like most other proteins, is asymmetric because of the

complex folding of its main chain.

An especially clear example of a metamorphic protein is the

cytokine lymphotactin.

When a protein is converted into a randomly coiled peptide without its normal activity, it is said to be

denatured.

The simplest sort of quaternary structure is a

dimer consisting of two identical subunits. This organization is present in Cro, a DNA-binding protein found in a bacterial virus called λ

Screw sense refers to the

direction in which a helical structure rotates with respect to its axis. If viewed down the axis of α helix, the chain turns in a clockwise direction; it has a right-handed screw sense. If turning is counterclockwise, the screw sense is left-handed.

In some proteins, the linear polypeptide chain is covalently cross-linked. The most common cross-links are

disulfide bonds, formed by the oxidation of a pair of cysteine residues. The resulting unit of two linked cysteines is called cystine. Disulfide bonds can form between cysteine residues in the same polypeptide chain or they can link two separate chains together. Rarely, nondisulfide cross-links derived from other side chains are present in proteins.

Lymphotactin

exists in two very different structures that are in equilibrium. One structure is a characteristic of chemokines, consisting of a three-stranded β sheet and a carboxyl-terminal helix. This structure binds to its receptor and activates it. The alternative structure is an identical dimer of all β sheets. When in this structure, lymphotactin binds to glycosaminglycan, a complex carbohydrate. The biochemical activities of each structure are mutually exclusive: the cytokine structure cannot bind the glycosaminoglycan, and the β-sheet structure cannot activate the receptor. Yet, remarkably, both activities are required for full biochemical activity of the cytokine.

The folding of proteins is sometimes visualized as a

folding funnel or energy landscape

Vitamin C is required for the

formation of stable collagen fibers because it assists in the formation of hydroxyproline from proline.

What can u find in the beta turn in the beta sheets?

glycine and proline

which two amino acids are usually not found in alpha helix

glycine and proline, bc they disrupt the function of the alpha helix,

The capacity of myoglobin to bind oxygen depends on the presence...

heme, a prosthetic (helper) group containing an iron atom.

dalton

is a unit of mass very nearly equal to that of a hydrogen atom.

The β pleated sheet

is almost fully extended rather than being tightly coiled as in the α helix. The distance between adjacent amino acids along a β strand is approximately 3.5 Å, in contrast with a distance of 1.5 Å along an α helix. The side chains of adjacent amino acids point in opposite directions

ariginie

is one of the nonessential but we need o get it n our diet still because we don't make enough of it for protein synthesis or metabolic requirements.

secondary structure is the

is the simple repeating structures formed by hydrogen bonds between hydrogen and oxygen atoms of the peptide backbone.

glycine

it isn't chiral, because it only has three different side groups since its R group is hydrogen. And because of this it doesn't have a D and L isomer.

In 1953, Frederick Sanger determined the amino acid sequence of insulin, a protein hormone (Figure 4.5). This work is a landmark in biochemistry because

it showed for the first time that a protein has a precisely defined amino acid sequence consisting only of L amino acids linked by peptide bonds.

A polypeptide chain has directionality because

its ends are different: an α-amino group is at one end, and an α-carboxyl group is at the other. By convention, the amino end is taken to be the beginning of a polypeptide chain, and so the sequence of amino acids in a polypeptide chain is written starting with the amino-terminal residue. Thus, in the pentapeptide Tyr-Gly-Gly-Phe-Leu (YGGFL), tyrosine is the amino-terminal (N-terminal) residue and leucine is the carboxyl-terminal (C-terminal) residue (Figure 4.2). The reverse sequence, Leu-Phe-Gly-Gly-Tyr (LFGGY), is a different pentapeptide, with different chemical properties

The formation of a dipeptide from two amino acids is accompanied by the

loss of a water molecule

A polypeptide chain consists of a regularly repeating part, called the... and a variable part, comprising....

main chain or backbone ( which is rich in hydrocarbon chains), the distinctive side chains.

What are two proteins, it has been known for some time that some proteins can adopt two different structures

metamorphic and intrinsically unstructured proteins.

domains can be made up of many

motfis

the helix turn helix is an example of a

motif... where several secondary structures form a structure found in many different proteins.

The largest protein known is the

muscle protein titin, which serves as a scaffold for the assembly of the contractile proteins of muscle. Titin consists of almost 27,000 amino acids.

Myoglobin interior consist mostly of

non polar residues.

Peptides made of small numbers of amino acids are called

oligopeptides or simply peptides.

motifs

or supersecondary structures, certain combinations of secondary structure are present in many proteins and frequently exhibit similar functions.

The importance of the positioning of glycine inside the triple helix is illustrated in the disorder

osteogenesis imperfecta, also known as brittle bone disease.

The linkage joining amino acids in a protein is called a

peptide bond (also called an amide bond)

The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds which are

phi and psi

A series of amino acids joined by peptide bonds form

polypeptide chain,

domains

polypeptide chains that fold into two or more compact regions that may be connected by a flexible segment of polypeptide chain, rather like pearls on a string. They range in size from about 30 to 400 amino acid residues. For example, the extracellular part of CD4, a cell-surface protein on certain cells of the immune system, comprises four similar domains of approximately 100 amino acids each (Figure 4.28). Different proteins may have domains in common even if their overall tertiary structures are different.

Proteins are complicated three-dimensional molecules, but their three-dimensional structure depends simply on their

primary structure—the linear polymers formed by linking the α-carboxyl group of one amino acid to the α-amino group of another amino acid.

Psi rotation

psi (ψ) is the angle of rotation about the bond between the carbonyl carbon and the α-carbon atoms.

each amino acid unit in a polypeptide is called a

residue

The essence of protein folding is the tendency to..

retain partly correct intermediates because they are slightly more stable than unfolded regions.

Most proteins have compact globular shapes, requiring reversals in the direction of their polypeptide chains. Many of these reversals are accomplished by common structural elements called....

reverse turns and loops. Loops exposed to an aqueous environment are usually composed of amino acids with hydrophilic R groups.

Then Anfinsen realized that once they were denatured and taken out of the urea and b-mercaptoethanol environment ...

ribonuclease slowly regained enzymatic activity. He immediately perceived the significance of this chance finding: the enzyme spontaneously refolded into a catalytically active form with all of the correct disulfide bonds re-forming. All the measured physical and chemical properties of the refolded enzyme were virtually identical with those of the native enzyme.

Less-stable collagen results in

scurvy

primary structure is the

sequence of amino acids

Cytokines are

signal molecules in the immune system that bind to receptor proteins on the surface of immune-system cells, instigating an immunological response.

tertiary structure, refers to the

spatial arrangement of amino acid residues that are far apart in the sequence and to the pattern of disulfide bonds. This level of structure is the result of interactions between the R groups of the peptide chain.

common feature of amyloidoses is

that normally soluble proteins are converted into insoluble fibrils rich in β sheets. The correctly folded protein is only marginally more stable than the incorrect form. But the incorrect forms aggregates, pulling more correct forms into the incorrect form.

Vitamin C is required for

the continued activity of prolyl hydroxylase, which synthesizes hydroxyproline. This reaction requires an Fe2+ ion to activate O2. This iron ion, embedded in prolyl hydroxylase, is susceptible to oxidation, which inactivates the enzyme. How is the enzyme made active again? Ascorbate (vitamin C) comes to the rescue by reducing the Fe3+ of the inactivated enzyme. Thus, ascorbate serves here as a specific antioxidant.

The depth of the funnel represents

the energy difference between the unfolded and the native protein.

What did Anfinsen experiment show us..

the information needed to specify the catalytically active three-dimensional structure of ribonuclease is contained in its amino acid sequence.

Collagen

the most-abundant mammalian protein. Collagen is the main fibrous component of skin, bone, tendon, cartilage, and teeth. It contains three helical polypeptide chains, each nearly 1000 residues long. Glycine appears at every third residue in the amino acid sequence, and the sequence glycine-proline-proline recurs frequently. Hydrogen bonds within each peptide chain are absent in this type of helix. Instead, the helices are stabilized by steric repulsion of the pyrrolidine rings of the proline residues (Figure 4.23).

hemoglobin

the oxygen-carrying protein in blood, consists of two subunits of one type (designated α) and two subunits of another type (designated β). Thus, the hemoglobin molecule exists as an α2β2 tetramer. Note that the hemoglobin subunits are called α and β for historical reasons, and the α and β designation has no relation to the α helix or the β strand.

What happened when the ribonuclease was treated with β-mercaptoethanol in 8 M urea?

the product was a randomly coiled polypeptide chain devoid of enzymatic activity.

peptide bonds are quite stable kinetically because

the rate of hydrolysis is extremely slow; the lifetime of a peptide bond in aqueous solution in the absence of a catalyst approaches 1000 years.

scurvy

the symptoms of scurvy include skin lesions and blood-vessel fragility. Most notable are bleeding gums, the loss of teeth, and periodontal infections. Gums are especially sensitive to a lack of vitamin C because the collagen in gums turns over rapidly.

This preference for trans over cis can be explained by the fact that

there are steric clashes between R groups in the cis configuration but not in the trans configuration

This freedom of rotation about two bonds of each amino acid allows proteins

to fold in many different ways.

the only polar residues on the interior are

two histidine residues, which play critical roles in binding the heme iron and oxygen. The outside of myoglobin, on the other hand, consists of both nonpolar and polar residues, which can interact with water and thus render the molecule water soluble. The space-filling model shows that there is very little empty space inside.

A β sheet is formed by linking

two or more β strands lying next to one another through hydrogen bonds. Adjacent chains in a β sheet can run in opposite directions (antiparallel β sheet) or in the same direction (parallel β sheet),

amino acids can twist and turn like beads on a string except

where the peptide bond is

Torsion angle

which is a measure of rotation about a bond, is usually taken to lie between −180 and +180 degrees. Torsion angles are sometimes called dihedral angles.

α-keratin

which is the primary component of wool and hair, consists of two right-handed α helices intertwined to form a type of left-handed superhelix called a coiled coil. α-Keratin is a member of a superfamily of proteins referred to as coiled-coil proteins

Special types of helices are present in two common fibrous proteins..

α-keratin and collagen.

A host of diseases are associated with improperly folded proteins such as ..

Alzheimer disease, Parkinson disease, Huntington disease, and transmissible spongiform encephalopathies (prion disease). All of these diseases result in the deposition of protein aggregates, called amyloid fibrils or plaques. These diseases are consequently referred to as amyloidoses

Most natural polypeptide chains contain between

50 and 2000 amino acid residues and are commonly referred to as proteins.

A protein with a molecular weight of 50,000 g mol−1 has a mass

50,000 daltons, or 50 kd (kilodaltons).