• Proteins (working molecules of a cell) carry out a program of activities encoded by genes

• Gradually many of the primitive proteins evolved into a wide array of enzymes cable of catalyzing an incredible range of intracellular and extracellular chemical reactions

• The functional design of proteins have “moving” parts and are capable of transmitting various forces and energy

3.1 - Hierarchical Structure of Proteins

• There are 20 different amino acids construed by polymerization into linear chains

• A protein is only able to function correctly when it is in its three-dimensional structure or conformation

The Primary Structure of a Protein Is Its Linear Arrangement of Amino Acids

• The primary structure of a protein is simply the linear arrangement or sequence of amino acids that residues compose

• Short-chain of amino acids linked by peptide bonds and having a defined sequence is called peptide

  • Fewer than 20-30 amino acids

• Long-chains are referred to as polypeptides

  • Can contain as many as 4000

• The term protein is usually reserved for polypeptides

• Proteins and peptides have been considered natural products of a cell

• The size of a protein (polypeptide) is represented by daltons

• Predicted proteins encoded by the yeast genome have an average molecular weight of 52.728 and contain an average of 466 amino acids

• The average molecular weight of amino acids in proteins is 113, taking into account their average relative abundance

Secondary Structure Are the Core Elements of Protein Architecture

• The second level in the hierarchy of protein structure consists of the various spatial arrangements resulting from the folding of localized parts of a polypeptide chain

  • These arrangements are referred to as secondary structures

• A single polypeptide may exhibit multiple types of secondary structure depending on its sequence

• The helix in a polypeptide segment folded into a helix

  • The carbonyl oxygen atom of each peptide bond is hydrogen-bonded to the amide hydrogen atom of the amino acid four residues toward the C-terminus

• This periodic arrangement of bonds confer a directionality on the helix because all the hydrogen-bond donors have the same orientation

• The hydrophobic or hydrophilic quality of the helix is determined entirely by the side chains

  • The polar groups of the peptide backbone are already engaged in hydrogen bonding in the helix

• The B Sheet is another type of secondary structure, the B sheet, consists of laterally packed B strands

• Each B strand is a short (5- to 8-residue), nearly fully extended polypeptide segment

• This structure is also called a B pleated sheet in a pleated sheet

  • Adjacent B strands can be oriented in the same (parallel) or opposite (antiparallel) directions with respect to each other

• Turns are composed of three or four residues, turns are located on the surface of a protein, forming sharp bends that redirect the polypeptide backbone back toward the interior

Overall Folding of a Polypeptide Chain Yields Its Tertiary Structure

• Tertiary Structure refers to the overall conformation of a polypeptide chain—that is, the three-dimensional arrangement of all its amino acid residues

• In contrast with secondary structures, which are stabilized by hydrogen bonds

  • Tertiary structure is primarily stabilized by hydrophobic interactions between the nonpolar side chains, hydrogen bonds between polar side chains, and peptide bonds

• These stabilizing forces hold elements of secondary structure— helices, strands, turns, and random coils—compactly together

• This variation in structure has important consequences on the function and

• regulation of proteins

• The simplest way to represent a three-dimensional structure is to trace the course of the backbone atoms with a solid line

• Another type of representation uses common shorthand symbols for depicting a secondary structure

• None of these three ways of representing protein structure convey much information about the protein surface

Motifs Are Regular Combinations of Secondary Structures

• Particular combinations of secondary structures, called motifs or folds, build up the tertiary structure of a protein

• Motif, also called the EF-hand, has been found in more than 100 calcium-binding proteins

• Many proteins, especially fibrous proteins, self-associate into oligomers by using a third motif, the coiled-coil

  • In these proteins, each polypeptide chain contains helical segments in which the hydrophobic residues, although apparently randomly arranged, are in a regular pattern (a repeated heptad sequence)

• The overall helical structure is amphipathic

Structural and Functional Domains Are Modules of Tertiary Structure

• The tertiary structure of proteins larger than 15,000 MW is typically subdivided into distinct regions called domains

• Large proteins, domains can be recognized in structures determined by x-ray crystallography or in images captured by electron microscopy

• A structural domain consists of 100–150 residues in various combinations of motifs

• Domains are sometimes defined in functional terms on the basis of observations that an activity of a protein is localized to a small region along its length

• DNA encoding a protein can be subjected to mutagenesis so that segments of the protein’s backbone are removed or changed

• The organization of large proteins into multiple domains illustrates the principle that complex molecules are built from simpler components

• The epidermal growth factor (EGF) domain is one example of a module that is present in several proteins

• EGF is a small, soluble peptide hormone that binds to cells in the embryo and in the skin and connective tissue in adults, causing them to divide

• EGF modules are also present in other proteins and are liberated by proteolysis

• Besides the EGF domain, these proteins contain domains found in other proteins

Proteins Associate into Multimeric Structures and Macromolecular Assemblies

• Multimeric proteins consist of two or more polypeptides or subunits

• The fourth level of structural organization, quaternary structure, describes the number (stoichiometry) and relative positions of the subunits in multimeric proteins

• Other multimeric proteins can be composed of any number of identical or different subunits

• Enzymes in the same pathway may be associated as subunits of a large multimeric protein within the cell, thereby increasing the efficiency of pathway operation

• The highest level of protein structure is the association of proteins into macromolecular assemblies

  • Such structures are very large, exceeding 1 mDa in mass, approaching 30–300nm in size, and containing tens to hundreds of polypeptide chains, as well as nucleic acids in some cases

• Macromolecular assemblies with a structural function

  • Include the capsid that encases the viral genome and bundles of cytoskeletal filaments that support and give shape to the plasma membrane

• The transcription factors and promoter-binding proteins correctly position a polymerase molecule at a promoter

  • The DNA site determines where transcription of a specific gene begins

Members of Protein Families Have a Common Evolutionary Ancestor

• Myoglobin and hemoglobin (the oxygen-carrying proteins in muscle and blood) respectively

  • Provided early evidence that function derives from three-dimensional structure, which in turn is specified by the amino acid sequence

• Subsequent sequencing of myoglobin and the hemoglobin subunits revealed that many identical or chemically similar residues are found in identical positions throughout the primary structures of both proteins

• Proteins that have a common ancestor are referred to as homologs

• The main evidence for homology among proteins

3.2 - Folding, Modification, and Degradation of Proteins

• A polypeptide chain is synthesized by a complex process called translation

• Incorrectly folded proteins usually lack biological activity and, in some cases, may actually be associated with disease

• Protein misfolding is suppressed by two distinct mechanisms

  • Cells have systems that reduce the chances for misfolded proteins to form

  • Any misfolded proteins that do form, as well as cytosolic proteins no longer needed by a cell, are degraded by a specialized cellular garbage disposal system

The Information for Protein Folding Is Encoded in the Sequence

• Any polypeptide chain containing n residues could, in principle, fold into 8n conformations

• Thermal energy from heat, extremes of pH that alter the charges on amino acid side chains

  • Chemicals such as urea or guanidine hydrochloride at concentrations of 6–8 M can disrupt the weak noncovalent interactions that stabilize the native conformation of a protein

• Sufficient information must be contained in the protein’s primary sequence to direct correct refolding

Folding of Proteins in Vivo Is Promoted by Chaperones

• Protein folding occurs in vitro, only a minority of unfolded molecules undergo complete folding into the native confirmation within a few minutes

• More than 95 percent of the proteins present within cells have been shown to be in their native conformation

  • Despite high protein concentrations (200–300 mg/ml), which favor the precipitation of proteins in vitro

• Molecular chaperones consist of Hsp70 and its homologs: Hsp70 in the cytosol and mitochondrial matrix, BiP in the endoplasmic reticulum, and DnaK in bacteria

• Molecular chaperones are thought to bind all nascent polypeptide chains as they are being synthesized on ribosomes

• In bacteria, 85 percent of the proteins are released from their chaperones and proceed to fold normally

  • An even higher percentage of proteins in eukaryotes follow this pathway

Many Proteins Undergo Chemical Modification of Amino Acid Residues

• Nearly every protein in a cell is chemically modified after its synthesis on a ribosome

• An important modification is the phosphorylation of serine, threonine, tyrosine, and histidine residues

• The side chains of asparagine, serine, and threonine are sites for glycosylation, the attachment of linear and branched carbohydrate chains

• Other post-translational modifications found in selected proteins include the hydroxylation of proline and lysine residues in collagen, the methylation of histidine residues in membrane receptors, and the -carboxylation of glutamate in prothrombin, an essential blood-clotting factor

Peptide Segments of Some Proteins are Removed After Synthesis

• After their synthesis, some proteins undergo irreversible changes that do not entail changes in individual amino acid residues

• This type of post-translational alteration is sometimes called processing

• An unusual and rare type of processing, termed protein self-splicing, takes place in bacteria and some eukaryotes

• This process is analogous to editing film: an internal segment of a polypeptide is removed and the ends of the polypeptide are rejoined

• Proteolytic processing, protein self-splicing is an autocatalytic process, which proceeds by itself without the participation of enzymes

Ubiquitin Marks Cytosolic Proteins for Degradation in Proteasomes

• The activity of a cellular protein depends on the amount present, which reflects the balance between its rate of synthesis and the rate of degradation in the cell

• The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins

  • Which help regulate passage through mitosis, to as long as the age of an organism for proteins in the lens of the eye

• Eukaryotic cells have several intracellular proteolytic pathways for degrading misfolded or denatured proteins

  • Normal proteins whose concentration must be decreased and extracellular proteins are taken up by the cell

• The major intracellular pathway is degradation by enzymes within lysosomes, membrane-limited organelles whose acidic interior is filled with hydrolytic enzymes

• The immune system also makes use of the ubiquitin-mediated pathway in the response to altered self-cells, particularly virus-infected cells

• Viral proteins within the cytosol of infected cells are ubiquitinated and then degraded in proteasomes specially designed for this role

Digestive Proteases Degrade Dietary Proteins

• The major extracellular pathway for protein degradation is the system of digestive proteases that breaks down ingested proteins into peptides and amino acids in the intestinal tract

• Three classes of proteases function indigestion

  • Endeoroteases

  • Exopeptidases

  • Peptidases

• To protect a cell from degrading itself, endoproteases and carboxypeptidases are synthesized and secreted as inactive forms (zymogens): pepsin by chief cells in the lining of the stomach; the others by pancreatic cells

Alternatively Folded Proteins Are Implicated in Slowly Developing Diseases

• Recent evidence suggests, that a protein may fold into an alternative three-dimensional structure as the result of mutations

  • Inappropriate post-translational modification, or other as-yet-unidentified reasons

• Such “misfolding” not only leads to a loss of the normal function of the protein but also marks it for proteolytic degradation

3.3 - Enzymes and the Chemical Work of Cells

• The function of nearly all proteins depends on their ability to bind other molecules, or ligands, with a high degree of specificity

Specificity and Addinity of Protein-Ligand Binding Depend on Molecular Complementarity

• Specificity refers to the ability of a protein to bind one molecule in preference to other molecules

• Affinity refers to the strength of binding

• The stronger the interaction between a protein and ligand, the lower the value of Kd

• For high-affinity and highly specific interactions to take place the shape and chemical surface of the binding site must be complementary to the ligand molecule

  • A property termed molecular complementarity

• The ability of proteins to distinguish different molecules is perhaps most highly developed in the blood proteins called antibodies

  • Which animals produce in response to antigens, such as infectious agents, and certain foreign substances

• The presence of an antigen causes an organism to make a large number of different antibody proteins

  • Each of which may bind to a slightly different region or epitope of the antigen

• Antibodies act as specific sensors for antigens, forming antibody-antigen complexes that initiate a cascade of protective reactions in cells of the immune system

• The specificity of antibodies is so precise that they can distinguish between the cells of individual members of a species and in some cases can distinguish between proteins that differ by only a single amino acid

Enzymes Are Highly Efficient and Specific Catalysts

• In contrast with antibodies, which bind and simply present their ligands to other components of the immune system, enzymes promote the chemical alteration of their ligands, called substrates

• Two striking properties of enzymes enable them to function as catalysts under the mild conditions present in cells: their enormous catalytic power and their high degree of specificity

• Approximately 3700 different types of enzymes, each of which catalyzes a single chemical reaction or set of closely related reactions, have been classified in the enzyme database

• Certain enzymes are found in the majority of cells because they catalyze the synthesis of common cellular products

• Other enzymes are present only in a particular type of cell because they catalyze chemical reactions unique to that cell type

• Most enzymes are located within cells, some are secreted and function in extracellular sites such as the blood, the lumen of the digestive tract, or even outside the organism

• The catalytic activity of some enzymes is critical to cellular processes other than the synthesis or degradation of molecules

An Enzyme’s Active Site Binds Substrates and Carries Out Catalysis

• Certain amino acid side chains of an enzyme are important in determining its specificity and catalytic power

• In the native conformation of an enzyme, these side chains are brought into proximity, forming the active site

• Active sites thus consist of two functionally important regions

  • One that recognizes and binds the substrate (or substrates)

  • One that catalyzes the reaction after the substrate has been bound

• For some enzymes, the catalytic region is part of the substrate-binding region; in others, the two regions are structurally as well as functionally distinct

• The active site of protein kinase A is located in the 240-residue “kinase core” of the catalytic subunit

• Substrate Binding by Protein Kinases; the structure of the ATP-binding site in the catalytic kinase core complements the structure of the nucleotide substrate

• The adenine ring of ATP sits snugly at the base of the cleft between the large and the small domains

• The catalytic core of protein kinase A exists in an “open” and “closed” conformation

• When the active site is occupied by the substrate, the domains move together into the closed position

• Phosphoryl Transfer by Protein Kinases; after substrates have bound and the catalytic core of protein kinase A has assumed the closed conformation, the phosphorylation of a serine or threonine residue on the target peptide can take place

Vmax and Km Characterize and Enzymatic Reaction

• The catalytic action of an enzyme on a given substrate can be described by two parameters: Vmax, the maximal velocity of the reaction at saturating substrate concentrations, and Km (the Michaelis constant), a measure of the affinity of an enzyme for its substrate

• The concentrations of the various small molecules in a cell vary widely, as do the Km values for the different enzymes that act on them

Enzymes in a Common Pathway Are Often Physically Associated with One Another

• Enzymes taking part in a common metabolic process are generally located in the same cellular compartment

• In the simplest such mechanism, polypeptides with different catalytic activities cluster closely together as subunits of a multimeric enzyme or assemble on a common “scaffold”

• In some cases, separate proteins have been fused together at the genetic level to create a single multi-domain, multi-functional enzyme

3.4 - Molecular Motors and the Mechanical Work of Cells

• A common property of all cells is motility, the ability to move in a specified direction

• Many cell processes exhibit some type of movement at either the molecular or the cellular level; all movements result from the application of a force

• Differently, materials within a cell are transported in specific directions and for longer distances

Molecular Motors Convert Energy into Motion

• At the nanoscale of cells and molecules, movement is affected by many different forces from those in the macroscopic world

• To generate the forces necessary for many cellular movements, cells depend on specialized enzymes commonly called motor proteins

• Motor proteins generate either linear or rotary motion

• This latter group comprises the myosins, kinesins, and dyneins, linear motor proteins that carry attached “cargo” with them as they proceed along either microfilaments or microtubules

• DNA and RNA polymerases also are linear motor proteins because they translocate along with DNA during replication and transcription

• The torque generated by the stator rotates an inner ring of proteins and the attached flagellum

• Interactions between the subunit and the B subunits directs the synthesis of ATP