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Isoelectric Points O R

+ H3N C CH NH COO- CH NH C If the side group is not ionizable, then the pI is the average of the CH C and N groups pKa R O R Calculations of pI for a compound with more than two dissociable groups carries more possibility for error Each amino acid is called a residue First write out all possible ionic structures for a compound in order that they occur starting with the most basic to the most acidic Addition of acid or base hydrolyzes the peptide Next, identify the isoionic, zwiterionic or neutral representation - The pI is the pH at the midpoint between the pK values on bond and adds water back across the peptide either side of the isoionic species bond.

What about peptides or proteins? The amino (N) terminal is written on the left and the carboxyl (C) terminal is on the right.

The Peptide Bond Peptide Bond

O R

H N COO- + 3 C CH NH CH NH C CH

R O R

– Rx of bond formation costs energy (ATP) but degradation of • has partial (40%) double bond character proteins is thermodynamically favorable. (entropy) • N partially positive; O partially negative – C-N bond shorter than normal and more like double bond • has a length of about 0.133 nm - shorter than a typical – R groups usually found in the trans conformation single bond but longer than a double bond – This results in rigid planar, non-rotating links between aa • Due to the double-bond character of the peptide bond, the – Size of peptides and proteins are described in Daltons six atoms of the peptide bond group define a plane – the – 1 atomic unit = 1 dalton ; MW = Dalton / kd; amide plane Mr=molecular weight

Fundamental Structural The Peptide Bond Pattern in Proteins

Size of R groups may favor/disfavor cis- peptide bond

The peptide bond is best described as a resonance hybrid The trans conformation of the forms shown on the two previous slides. of the peptide bond. The cis-peptide bond

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Fundamental Structural Peptide Backbone Pattern in Proteins Rotatable peptide bonds • Due to peptide bond (review) • Trans conformation of a carbon is more stable and primary conformation • Consider the consequence of this arraignment • 10% of Pro are in Cis.

Peptide Backbone

Limited rotation around a carbon • Dihedral/torsion angles describe the rotation around the a carbon • C-N Phi and C-C Psi • The peptide bond is creates limited possibilities. • Torsion angles are defined by 4 points or planes • Notice the side groups When proline is in a peptide bond, it does not have a hydrogen on the α amino group, so it cannot donate a hydrogen bond to stabilize an α helix or a β sheet.

Many peptides have important Peptide Backbone biological activities

Limited Options Insulin - Short peptide produced as a pre-pro-peptide. The • Peptide bond limits rotation along all initial peptide is much longer and is modified twice, each time bonds in backbone creating a a set of peptide bonds are hydrolyzed. The final shortened • 3D spatial arrangement is determined version is active. There are two subunits, held together by a by the limited rotational options disulfide bond. • R groups add limitations – steric • Amide H and Carbonyl O also impact rotation options • R groups have NOTHING to do with peptide bond issue

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Structure of Insulin Insulin

Primary structure tells of relatedness

The inactive insulin is a hexamer, where via processing it loses the metal ion and is converted to an active monomer

Differences of one or two amino acids make the peptide more immunoreactive (allergenic) to some

Change affinity for Gluten – a tale of two receptor proteins

Glutenin – Long and very large proteins which have lots of sulfur atoms. Coiled proteins which can stretch and recoil. Sulfur

Long-Acting Fast -Acting (cystine side chains) help hold these together Gliadins – Smaller proteins which act like Fast-acting insulin is usually taken just after or before meals for controlling blood sugar spikes. It often begins to work in 10 to 15 lubricants or ball bearings – allowing parts of minutes, peaks in about 1 hour, and lasts for about 3 to 5 hours. glutenins to move past each other Long-acting insulin begins action after 1 to 2 hours. Its insulin effect plateaus for the next few hours and then a relatively flat action duration follows, and this lasts for about 15 to 24 hours.

Dough Strength = Gluten Formation

Glialdin Glutenin

Raw un-worked dough Stretched and kneaded dough Loose structure – no gluten Elastic and strong structure = gluten Coils and sulfur cross-links (black lines) of glutenin give the elastic ability to dough • High protein (and gluten) flour give strong gas pockets good for breads • Low gluten flours are weaker tender baked goods – cakes and cookies (that is why you don’t over mix pancake mix) • High water dough (called batter) dilutes gluten so they don’t mix/bind - Click here to explore Gluten

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Amino Acids Give Why so many Glutamines? Function

Glutamine supplies N for growing seedling. ALSO Glutamine side chains (amine) can hydrogen bond with another glutamine forming a crosslink between strands of glutenin • If there were less glutamines, the amine The protein sequence in single letter amino acid would bind to water abbreviations for glutenin. Notice the high proportion of instead reducing Q & P (highlighted in dark shading) and the low cross-linked strength abundance of charged D & E (Shaded in light color)

Proline What about Cysteines?

Up to 50% of amino acids of glutenin can be proline – typical protein is ten to 20 times lower • Prolines are the most inflexible amino acids and in a peptide / protein backbone lead to bends and kinks Two cysteine amino acids bond forming a disulfide – a cross- breaking up helixes started by glutamine linked glutenin is more stable and mechanically rigid • This gives glutenin its elasticity

Strengthen your flour Bioactive Peptides muscle Oxytocin and Vasopressin – Start as long precursors in hypothalamus- whose final form is 9 aa Oxidizing agents – alter sulfur links and increases – differ by 2 residues • oxytocin - uterine contraction during childbirth and milk production strengths of gluten during lactation • Flowing oxygen gas through the flour “ages” it by • vasopressin - alters blood pressure by forcing kidney to retain water, allowing sulfur-sulfur links to form – giving more increasing the volume of blood cross-links (stronger flour) Met-enkephalin (opioid peptides) • Chlorine gas and brominates (can do same thing) but » Naturally produced peptides, bind to receptors, and reduce no longer approved pain cause pleasure. Morphine like • Ascorbic acid (vitamin C) is now used instead of Substance P » Opposite effect from opioids gasses. » Stimulates perception of pain (protective mechanism) • Also causes the flour to whiten (bleaching)

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Proteins: Three- dimensional structure Cofactors Defined

Background on protein composition: Cofactors are nonprotein Two general classes of proteins groups required for protein • Fibrous - long rod-shaped, insoluble proteins. These proteins are strong binding or activity. (high tensile strength). Examples: keratin, hair, collagen, skin nails etc… • Globular - compact spherical shaped proteins usually water-soluble. Most – Can be divided into two hydrophobic amino acids found in the interior away from the water. Nearly categories: minerals all enzymes are globular… an example is hemoglobin (metal ions) and vitamins Proteins can be simple - no added groups or modifications, just amino acids (small organic groups) – Metal ions (Ca2+, Mg2+, Or proteins can be conjugated. Additional groups covalently bound to the Zn2+, Fe2+, and Cu2+) amino acids. The naked protein is called the apoprotein and the added group is the prosthetic group. Together the protein and prosthetic group is – Organic groups (NAD+ called the holoprotein. Ex. Hemoglobin and FAD, pyridoxal phosphate)

Levels of Structure Primary Structure

Type of Structure: Defines: Type of Bonds: Primary (1°) Order of amino acids Covalent bonds Secondary (2°) Local structure Non-covalent The order of the 20 common amino acids is the primary (α-helix, β-sheet, loop) interactions, disulfides structure Tertiary (3°) Overall fold, 2° Non-covalent elements organize and interactions, disulfides - shape, orientation and other information is not part of the compact—low surface primary structure to volume ratio - Primary structure is informative for determining - Relativeness to other proteins or regions of proteins Quaternary (4°) Subunit organization, Non-covalent - Predict structure dimers, macro- interactions, disulfides - Determine domain (group of aa that has a function) molecular assembly - Predict RNA and DNA sequence

Secondary Structure α-helix

Primary Secondary/Tertiary FUNCTION! Discovered by Linus Pauling Structure Structure •2 Nobel prizes. •Discovered folding while sick in bed! Protein structure is defined by the •Treated poorly as a result of his anti-nuclear nd peptide plane and the 2 degrees of stands - 2 NB prize freedom around the peptide bond • The helix is a right handed twist of the backbone - Two basic predictable forms of notice when we are looking at this the side groups secondary structure are NOT considered Alpha helix and beta pleated sheet • Notice where the amino acids are. • Hydrogen bonding occurs between the carbonyl -Other forms are specialized and the amino group ~four residues away (3.6 aa/turn). The bonding takes place within the same (collagen) chain. i+4 - Beta sheets, tight turns and beta bulge A run of proline residues lead to breaking the helix structure. Why?

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α-helix α-helix

Formation of α-helices are governed by HYDROGEN For an a-helix of n residues, BONDS! One helix turn is 3.6 amino there are n-4 hydrogen acid residues, and involves bonds! 13 atoms from the O to the H Helix capping—the last 4 of the H bond amide hydrogens/carbonyl 13 oxygens cannot H-bond. For α-helix: Proteins compensate by Pitch: 5.4Å 1 ϕ ~60° folding other parts of the Ψ ~-45 – -50° protein to facilitate hydrogen bonding.

Diameter (w/o Side Chains): ~6Å

α-helices are polar! β-Pleated Sheet

H-bonds all point in the same Neg. charge, Notice that direction Rarely find pos. there are no Amide bond has a dipole ligands. “turns” pictured moment—cumulatively, the helix here—the sheet has a large dipole moment Why? here is not a contiguous amino acid + charge, sequence! Binds neg charged Side chains ligands perpendicular to (e.g. plane of the Phosphates) sheet Defined by a different hydrogen-bonding network Note: hydrogen bonds occur interstrand

Parallel and Anti-Parallel β- Sheets Hairpin (tight) turns and loops

Secondary structures are connected by turns and loops Parallel: Adjacent chains run in the same direction • Bent H-bonds

• Normally large, >5 strands 0.325 nm 9 different types of tight hairpin • Hydrophobic side chains loops distributed on both sides of sheet Anti-Parallel: Adjacent chains run Defined by torsion angles in the opposite direction Type 1 β-Turn • More extended H-bond conformation Tight turns – 3-4 aa • Can consist of 2 strands Loops or coils 5/6 and greater

• Hydrophobic on same side— 0.347 nm alternates hydrophilic and hydrophobic in primary sequence

Let’s go back to the previous slide—what kind of sheet?

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