Introduction to Proteins

Introduction to Proteins

• Proteins have varied functions arising from key structural features.

Linear Polymers of Amino Acids

• An unlimited order of amino acids allows for a vast variety of proteins
• Each protein is a biologically functional, unique sequence of amino acid residues folded into a unique and stable 3D structure
• These amino acids are linked by stable peptide bonds, and peptide bonds can last thousands of years without an enzyme

Functional Groups in Proteins

• Proteins contain a wide range of functional groups, including hydroxides, thiols, thioesters, carboxylic acids, amines, and phosphates
• The reactivity of these functional groups accounts for enzyme activity

Protein Interactions and Assemblies

• Proteins interact with each other and other biological molecules to form complex assemblies
• Molecular cooperation leads to molecular synergism

Polypeptide Chains

• A protein may consist of a single polypeptide chain or many chains

Conformation Importance

• 3D shape is critical to the biological processes of proteins
• The spatial arrangement of atoms that depends on rotation of bonds defines protein conformation
• Conformation does not break covalent bonds
• Polypeptides have numerous possible conformations.
• Native conformation is the single, stable shape a protein assumes under physiological conditions.
• A protein's biological function depends completely on its conformation

Genome Encoding

• The number of polypeptides encoded in a genome can be determined through genomic sequencing
• E. coli contains approximately 4,000 polypeptides
• D. melanogaster contains approximately 14,000 polypeptides.
• H. sapiens contains roughly 20,000 different polypeptides.
• Proteomics is the study of the entire complement of proteins in a cell

Protein Rigidity and Flexibility

• Some proteins are rigid, while others exhibit limited flexibility
• Rigid proteins are often structural proteins
• Flexibility is useful in making hinges, springs, and levers

Globular vs. Fibrous Proteins

• Proteins can be classified as either globular or fibrous, based on their structure
• Fibrinogen is a fibrous protein
• Superoxide dismutase exemplifies a globular protein

Primary Structure

• This is the linear sequence of amino acids held together by covalent forces (peptide bonds).
• The primary structure determines the overall shape of folded polypeptides and influences secondary, tertiary, and quaternary structures

Secondary Structure

• This element involves local regions with repeating conformations like alpha helices, beta sheets, and loops
• Secondary structures are maintained by hydrogen bonds (H-bonds) between amide hydrogens and carbonyl oxygen
• Examples include alpha helix and beta sheets

Tertiary structure

• The completely folded and compacted polypeptide chain represents the tertiary structure.
• It is stabilized by the interactions of side chains of non-neighboring amino acid residues

Quaternary Structure

• This arrangement involves the association of two or more polypeptide chains into a multi-subunit protein

Protein Sequencing

• Primary structure is determined through DNA or sequencing

Determining 3-D Structure

• Elucidating 3-D native conformation is crucial for understanding protein function
• X-ray crystallography and nuclear magnetic resonance (NMR) imaging can determine 3-D conformation

X-Ray Crystallography Process

• A beam of parallel X-rays is directed at a protein crystal
• Electrons in the crystal diffract the X-rays
• The diffraction of X-rays by electrons is recorded on film or by an electronic detector
• Mathematical analysis of the diffraction pattern, along with knowledge of the primary structure, is used to deduce 3-D conformation
• The development of computers has aided this process

Max Perutz

• Determined the structure of hemoglobin

John Kendrew

• Determined the structure of myoglobin

Nobel Prize

• Perutz and Kendrew shared the Nobel Prize in 1962

Pros of X-Ray Crystallography

• Proteins retain their native conformation in crystal form
• Crystals contain 20-50% water molecules
• Researchers can add ligands, which diffuse into active sites
• Crystals retain catalytic activities, which shows the crystal represents active, in vivo conformation

Cons of X-Ray Crystallography

• Requires preparation of protein crystals
• It can be difficult and time-consuming
• Some proteins do not easily adopt a crystalline form

NMR Spectroscopy

• A sample of protein is placed in a magnetic field
• Electromagnetic radiation is applied, and certain atomic nuclei absorb it as the applied magnetic field varies
• Absorbance reveals interactions of atoms that are close together
• Data is combined with knowledge of the amino acid (aa) sequence to calculate a number of structures that satisfy the observed interactions

Bovine Ribonuclease A

• An NMR structure

Pros of NMR Spectroscopy

• Permits study of proteins in solution
• Does not require preparations of crystals

Cons of NMR Spectroscopy

• Better suited for use for small proteins
• Difficult to determine the structures of large, complex proteins

Discovery of the Alpha Helix

• Discovered by Linus Pauling in 1950
• This structure is the most frequently observed secondary structure
• The structure is virtually always found in nature as right-handed form

Alpha Helix Details

• An alpha helix generally has ~3.6 (3.5-3.7) residues per turn
• Pitch (advance per turn) is 0.54 nm
• Rise (advance per amino acid residue) is 0.15 nm

Alpha Helix Bonding and Location

• Each carbonyl oxygen (residue n) of the polypeptide backbone is H-bonded to the backbone amide hydrogen of the fourth residue toward the C-terminus (n + 4)
• The first three and last three residues lack H-bonding partners within the helix
• Each hydrogen bond closes a loop of 13 atoms
• Hydrogen bonds are nearly parallel to the long axis of the helix
• All carbonyl groups point toward the C-terminus
• Stabilized by hydrophobic regions.

Side Chain Impact

• The side chains of amino acids in an alpha helix point outwards from the cylinder of the helix
• Alanine, with its small, uncharged side chain, fits well within the helix
• It is often found in helices of globular proteins
• Tyrosine and asparagine are less common due to their bulky R-groups
• Glycine destabilizes helices because rotation around the alpha-carbon is unconstrained, disrupting the normal structure/rigidity of the peptide backbone
• Proline is never found in the interior of alpha-helices, due to the rigid cyclic side chain causing steric interference
• Proline also lacks amide hydrogen for H-bonding with carbonyl oxygen of other amino acids in the helix.

Termination

• Alpha helices usually end with structures known as the helix stop signal (or capping box)
• The helix stop signal is a specific pattern of amino acid residues at the beginning of an alpha helix
• The side chain of N-terminal residue (n) of helix interacts with backbone amide hydrogen of the fourth residue of helix, resulting in a conformation that is not compatible with formation of alpha helix
• Serine and threonine are usually found at initial positioning of the capping box, and glutamate is often the fourth residue

Alpha Helix Content

• Globular proteins vary in their alpha-helical content
• Myoglobin contains 75% alpha-helices
• Chymotrypsin has very little
• Average for globular proteins is ~26%

Amphipathic Helices

• These structures have hydrophilic amino acids on one face of the helix cylinder and hydrophobic amino acids on the opposite face
• They are often located on the surface of globular proteins

Beta Structure

• Portions of the polypeptide chain are almost fully extended in the beta structure
• It includes beta strands and beta sheets
• Beta sheets consist of multiple beta strands arranged in sheets
• Stabilized by H-bonding between carbonyl oxygens and amide hydrogens on adjacent beta strands (of same or different polypeptide)

Parallel vs. Antiparallel Beta Strands

• In beta sheets, side chains point alternately above and below the plane of the sheet
• Parallel strands are less stable because of disordered H-bonds
• Beta sheets may contain 2-15 strands and can form barrel-shaped structures

Beta-Sandwich

• Two antiparallel sheets stacked against each other
• One side of the sheet is hydrophilic, while the other is hydrophobic
• These sheets come together so that hydrophobic regions face each other in the center of the protein
• Hydrophilic regions face the surface

Random Coils/Loops and Turns

• These regions are non-repetitive, but are actually stable, highly-ordered structures
• They connect secondary structures and provide directional changes necessary for proper folding
• Examples include loops, turns, and hairpin loops
• Globular proteins can contain as many residues in non-repetitive conformations as in helices and strands
• The least conserved areas of protein e.g. in the variable domains of the antibody molecule that bind to antigens

Motifs

• Supersecondary structures
• Combinations of alpha helices and beta strands that appear in a number of different proteins can be recognized
• These motifs sometimes have a conserved function between different proteins

Tertiary Structure

• This structure results from the folding of a polypeptide into a closely packed, spherical shape
• Non-covalent interactions (mostly hydrophobic effects) between amino acid and side chains primarily stabilize the whole structure
• There is great variety in tertiary structures

Protein Domains

• Many globular proteins are comprised of independently folded globular units called domains.
• Combinations of motifs makeup domains, and these range in size from 30-300 amino acids
• Small proteins usually contain one domain
• Larger proteins may contain more than one domain
• Often each domain has a function
• In multifunctional enzymes, each catalytic activity is associated with a different domain
• Regions between domains form crevices, which sometimes serve as binding sites
• Some domain structures occur in many different proteins

Quaternary Structure

• This structure is limited to proteins with multiple subunits
• Each subunit is called a monomer
• A multisubunit protein is called an oligomer
• Subunits in an oligomeric protein always have a definite stoichiometry
• Monomers may be identical or different
• Dimers and tetramers are the most common when monomers are identical
• When the monomers differ, each subunit has a unique function
• When several metabolic reactions are catalyzed by an oligomer, it is called a multienzyme complex
• Oligomer subunits are usually held together by noncovalent forces - hydrophobic interactions

Purpose of Quaternary Structure

• More stable than dissociated subunits
• Active sites of some oligomers are formed by amino acids on adjacent polypeptides
• Likelihood of errors is greater for long polypeptides
• It is more efficient to synthesize several smaller subunits
• 3D-structure changes when proteins bind ligands - critical step in biological activity of many proteins
• Different proteins can share the same subunits
• Allows for sequential enzyme action

Protein Folding Process

• As it is translated, the peptide begins to fold
• The first few interactions begin subsequent interactions by assisting in the alignment of groups
• Cooperative folding refers to when the formation of one part of a structure leads to the formation of the remaining parts
• Protein folding and stabilization depend on non-covalent forces - hydrophobic effect, H-bonding, charge-charge interactions, and van der Waals forces
• Formation of disulfide bonds (covalent)

Native Conformation and Stability

• The native conformation of a protein is its most stable shape
• Protein folding is a spontaneous process
• The process is driven by an overall increase in entropy coupled with a decrease in free energy
• Sections of polar backbone that are forced into interior neutralized by H- bonding

Chaperones

• Chaperons assist in folding by ensuring the correct folding pattern
• Some proteins require chaperones in order to fold correctly
• Chaperones prevent incorrectly folded intermediates from arising in the first place
• Chaperone do not "direct" folding; rather, they make sure that the target proetin achieves maximum stability

Denaturation

• Denaturation refers to disruption in the native conformation of a protein, resulting in a loss of biological activity
• Altering very few interactions is sufficient to denature proteins

Protein Denaturation Via Heating

• Increased thermal energy causes more rapid vibrations of molecules
• Heating can break relatively weak hydrogen bonds and hydrophobic interactions

Protein Denaturation Via Changes in pH

• Acids and bases alter the ionization of amino acid side chains
• This alters/disrupts ionic bonds/salt bridges that stabilize protein structure

Protein Denaturation Via Chaotropic agents

• Molecules that can disrupt the hydrogen bonding network between water molecules ultimately disrupting hydrophobic interactions
• These agents compete for H-bonding partners within polypeptide backbones and side chains disrupting H-bonds stabilizing protein structures

Protein Denaturation Via Detergents

• Compete for ionic bonds partners and/or non-polar side chains in proteins
• Disrupts interactions that stabilize protein structures

Protein Denaturation Via Cleavage

• Breaking disulfide bonds using mercaptoethanol, a reducing agent

Ribonuclease

• Scientists have demonstrated that correct disulfide bonds only form after proteins fold into native conformation

Collagen

• A common fibrous protein that is 25-30% of total protein in animals
• Made by connective tissue cells and secreted to the extracellular matrix
• Gives high tensile strength to connective tissue, as in teeth, bones, skin, tendons, ligaments, blood vessels, and cartilage
• There exists a diverse form and function
• The collagen in tendons are stiff, rope-like fibers
• The collagen in skin creates loosely woven fibers that permit expansion in all directions

Collagen Composition

• Consists of 35% Gly, 11% Ala, 21% Pro & Hyp (hydroxyproline)
• Its primary structure consists of repeating tripeptide Gly-X-Y with e.g. Gly-Pro-Hyp
• Collagen fibrils consist of 3 left- handed helical polypeptides (alpha chains) wrapped into a right-handed triple helix (superhelix or tropocollagen)
• They are arranged head-to-head in parallel bundles.

Tropocollagen

• Covalent cross-links between molecules of tropocollagen are responsible for that rigidity and tensile strength of collagen fibers

Collagen Structure

• Collagen triple helix is stabilized by inter-chain H-bonds
• Glycine residues in each form H-bonds with X residue of adjacent chain
• No intrachain H-bonds in collagen helix

Vitamin C

• Hydroxylation of proline and lysine catalyzed by enzymes often require ascorbic acid (vitamin C)
• Vitamin C deficiency results in improper formation of collagen resulting in scurvy - Skin lesions, bleeding gums, loose teeth, fragile blood vessels, etc.

Myoglobin Function

• Myoglobin functions by selectively and reversibly binding to O2 in muscles
• It is a globular protein that contains a heme prothetic group
• The heme prosthetic group gives source of red color in muscles and blood
• The protein is a member of globin multigene family, which also includes hemoglobins and leghemoglobin from plants
• High concentrations of myoglobin in muscle cells allow organisms to hold their breath for a longer period of time
• Diving mammals such as whales and seals have muscles with particularly high abundance of myoglobin

Myoglobin Structure

• Consists of 8 alpha-helices
• Contains heme prosthetic group
• Protein-bound organic molecule
• Protein molecule activity depends essential
• Heme molecule: complexed to iron (Fe2+)
• Forms a complex with six ligands: 4 N of protoporphyrin ring, His-93, and O2

Heme Prosthetic Group

• Prosthetic group of myoglobin wedged into hydrophobic cleft
• No covalent bonds hold heme group
• Protein structure: essential for the reversible binding of oxygen
• Oxidation of heme prevents reversible interaction with oxygen

Hemoglobin Functions

• Functions by selectively and reversibly binding O2 in erythrocytes
• Transports O2 in the blood from gas-exchange organs to tissues
• Has 300 million/erythrocyte and is globular/tetrameric
• Is oligomeric A2B2 , with alpha and beta chains that are similar and each have an identical heme prosthetic group
• Unique oxygen-binding properties compared to myoglobin
• Acts as a dimer of alpha/ beta subunits

Oxygen-Binding and Saturation

• The extent of oxygen binding at equilibrium depends on the concentration of the protein and the concentration of oxygen
• Fraction of molecules that are oxygenated (Y) vs. partial pressure of gaseous oxygen (pO2)
• Y = % saturation
• Oxygen-binding curves help determine the relationship between myoglobin and hemoglobin

Hyperbolic vs Sigmoidal

• Myoglobin has a hyperbolic oxygen binding curve, while hemoglobin has a sigmoidal O2- binding curve
• Hyperbolic curves are indicative of single ligand binding
• Sigmoidal is indicative of more than ligand binding
• Each tetramer can bind up to 4 O2, 1 per heme group

Cooperative Binding

• Shape of curve suggests that binding of first O2 facilitates binding of O2 to other heme molecules
• The oxygen affinity of hemoglobin increases as each O2 is bound
• Illustrates positive cooperative binding

Hemoglobin States

• Cooperative binding is related to changes in protein confirmation
• Tense conformation- Deoxyhemoglobin/Low oxygen affinity
• Movement disrupts ion pairs within the protein
• Favors conformation with higher oxygen affinity
• RelaxedConformation-Oxyhemoglobin/High oxygen affinity

Oxygen Affinity

• The partial pressure at half-saturation (P50) is the measure of the affinity of the protein for O2

Globin Protein Binding Systems

• Differential affinities for O2 separate myoglobin and hemoglobin in terms of oxygen delivery
• At high po₂ the proteins become saturated
• At low pO2, hemoglobin loses O2
• Myogobin then picks up 02

Allosteric Protein

• Hemoglobin is an allosteric protein that contains 2,3-bisphospho-D-glycerate (BPG)
• BPG is a by-product of glycolysis and is produced by metabolically active tissues
• Binding of BPG to hemoglobin reduces affinity of hemoglobin to O2 by lowering its capacity to bind oxygen

Regulators of O2 Affinity

• Both H+ and CO2 also affects affinity of hemoglobin for O2
• In the Bohr Effect, this system affects the conformation of said protein

Regulation Summary

• An increased amount of H+/CO results in in a lower pH
• Both of these compounds lower the resulting overall affinity

High/Low Metabolism

• High metabolism=Reduced 02 affinity
• Low metabolism= Increased 02 affinity