BIOCHEMISTRY BLOCK 1

Amino Acid Distribution in Porins (Tertiary)

"Inside Out" Hydrophilic lined water channel Largely hydrophobic exterior

pI =

(pKr + pK1) / 2

pH increased:

+ on the NH3 removed AA will move to anode

pK =

-log Keq

To break a polypeptide chain requires extreme conditions such as:

1) High acid 2) High temperature *Necessary to break series of strong covalent bonds *Called Chaotropic reagents

Derived Amino Acids:

After incorporation into proteins, some amino acids are enzymatically modified.

Trypsin will cleave at:

Arg, Lys

In mammals, the ___________ is the major extracellular buffer system.

Carbonic acid-bicarbonate (CO2/HCO3-) CO2 + H20 -> H2CO3 -> HCO3- + H+

What is the method for studying secondary structure?

Circular dichroism (CD) Spectroscopy

Examples of fibrous proteins:

Collagen Keratin Tropomyosin

Why are proteins important?

Defense (Antibodies) Structure (Collagen) Transport (Hemoglobin) Catalysis (Cytochrome oxidase) Movement (Myosin) Regulation (Insulin) Storage (Ferritin) Signaling (Rhodopsin)

_______ causes a loss of secondary, tertiary, and quaternary structures due to unfolding of the polypeptide chain. A denatured protein is incapable of carrying out its biochemical and biological functions.

Denaturation

You have isolated a tripeptide and do not want to carry out amino acid analysis. You determine that the peptide contains sulfur, absorbs UV light at 280 nm, and is cleaved by trypsin. Which of the following is consistent with these properties? A. Trp-Lys-Ser B. Tyr-Phe-His C. Met-Trp-Thr D. Lys-Ser-Cys E. Trp-Lys-Met

E

Metabolic Acidosis

Excess organic acids like in Diabetes (ketone bodies) or hypoxemia (lactic acid) or loss of HCO3- (severe diarrhea, uremia, CKD)

Collagen is the major insoluble fibrous protein in the _______

Extracellular matrix and in connective tissue -It is present in all tissues and organs and provides the framework that gives the tissues their form and strength

Unique Side Chain Amino Acids

Glycine (Gly, G) Proline (Pro, P)

pH decreased:

H+ added to COO AA will move to cathode

Dissociation of weak acid HA releases a proton H+

HA -> H+ + A-

Protein buffer system

Hemoglobin also acts as a pH buffer in the blood as the hemoglobin protein can REVERSIBLY bind either H+ (to the protein) or O2 (to the Fe of the heme group). *When one of these substances is bound, the other is released (exchange system).

Types of Quaternary Structure:

Homodimer (a2) Heterodimer (ab) Heterotetramer (a2b2) Heteropentamer (a2bcd)

Respiratory Aklaosis

Hyperventilation (Hysteria, fever, drugs)

Acid (proton donor) _______ concentration of H+ in an aqueous solution

Increases

Only the ___ configuration of proteins is found in nature

L

Amino Acids were first discovered by French chemists ________ and ______ in 1806.

Louis-Nicolas Vauquelin Pierre Jean Robiquet

Bromelain will cleave at:

Lys, Ala, Tyr, Gly

LECTURE 2

PEPTIDES & PROTEIN

Chymotrypsin will cleave at:

Phe, Tyr, Trp

Aromatic (Hydrophobic) Amino Acids

Phenylalanine (Phe, F) Tyrosine (Tyr, Y) Tryptophan (Trp, W)

20 common amino acids which are _______ in nature.

Proteinogenic

_____ perform functions in every system and are essential for physiological processes

Proteins

Pyrrolysine

Pyr, O Weak Basic *Present in Archaea, Bacteria

Enzymes cleave at which terminus?

The C-terminus side (ie. right side)

Tertiary Structure:

The folded conformation of a protein, formed by the condensation of various secondary elements, stabilized by a large number of weak interactions (hydrophobic interactions, H bonds, and salt bridges)

Absorption spectra of _____ and ____ is strongest around 280nm

Trp and Tyr

What is the best buffering range?

When pK = pH When base to acid = 1

What is the strongest bond?

covalent

Secondary Structure:

folded segments of a polypeptide chain with repeating, characteristic torsion angles, that are stabilized by a regular pattern of H-bonds between the peptide NH and CO groups of different residues

B-Pleated Sheet

formed by backbone H bonding between segments of extended polypeptide chain

Hydrophobic amino acids are located _____

inside of folded proteins

Amino Acid Functions:

1) Acid/Base Homeostasis 2) Neurotransmitters 3) Precursors for hormones 4) Protein synthesis 5) Cell signaling 6) Chemical messengers 7) Metabolic regulation

Steps in Sequencing a Polypeptide:

1) Determination of amino acid composition 2) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide.

Proteins Provide:

1) Energy 2) Maintenance (Growth, Structural) 3) Regulatory (Enzymes, Gene Expression, Hormones, Fluid Balance, Transport, Acid/Base Balance, Antibodies)

Rare Amino Acids

21st amino acid: Selenocysteine (Sec) 22nd amino acid: Pyrrolysine (Pyl) *Encoded by UGA (Sec) and UAG (Pyl), which normally serve as STOP codons, in presence of specific insertion sequence elements *The tRNA is charged with standard amino acid and then enzymatically modified

Aliphatic (Hydrophobic) Amino Acids

Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L) Isoleucine (Ile, I)

______ was the first amino acid to be discovered.

Asparagine (after their favorite food asparagus)

Body Fluid pH's

Blood plasma (7.4) Interstitial Fluid (7.4) Gastric Juice (1.3-3.0) Pancreatic Juice (7.8-8.0) Human Milk (7.4) Saliva (6.4-7.0) Urine (5.0-8.0)

Keq is a constant based on concentration and is determined by the equation:

Keq = (H)(A)/(HA) ***Concentration of each component in M

CNBr will cleave at:

Met

Respiratory Acidosis

Retention of CO2 (Asthma, causing a decrease in exhalation of CO2)

Metabolic Alkalosis

Retention of HCO3- (ingestion)

Selenocysteine

Sec, U Acidic *Present in Archaea, Bacteria, and animals (including mammals)

Proteins have a repeating ______ from which 20 different possible kinds of side chains protrude. On rare ocassions, nonstandard side chains are found.

backbone

Salt Bridge:

A hydrogen bond in which both donor and acceptor atoms are permanently fully charged. The bonding energy of a salt bridge is significantly higher than that of a Hydrogen bond in which only one participating atom is fully charged or in which both are partially charged.

Normal metabolism is associated with a continuous production of hydrogen ions (H+) and CO2. These can _________ which has deleterious effects including but not limited to: reduced oxygen delivery to tissues, electrolyte disturbances, and changes in heart muscle contractility.

Reduce pH *Maintain normal pH -> Preserve acid-base homeostasis. Kidneys and lungs work together to help maintain a blood pH of 7.4 by affecting the components of buffers in the blood.

Codon

Sequence of 3 nucleotides which specifies the amino acid

Large Ka means

Strong acid

Proteins are made of amino acids which are strung together in order by a _______

covalent peptide bond

Alkalosis

pH of blood rises above 7.45

Fibrous Proteins

typically contain larger amounts of regular secondary structures than globular proteins -long cylindrical (rod-like) shape -LOW solubility in water -A structural rather than a dynamic role.

Acidic Amino Acids

Aspartate (Asp, D) Glutamate (Glu, E)

Zwitterion

At this pH, it is at its isoelectric point (carries no net electric charge) NH3+ COO-

Acid hydrolysis of a peptide reveals equimolar amounts of Lys, Gly and Ala. Following trypsin digestion of the peptide, only free Gly and a dipeptide are observed upon analysis. Which of the following sequences is consistent with these properties? A. Gly-Lys-Ala-Lys-Gly-Ala B. Ala-Lys-Gly C. Lys-Gly-Ala D. Gly-Lys-Ala E. Ala-Gly-Lys

B

Basic Amino Acids

Lysine (Lys, K) Arginine (Arg, R) Histidine (His, H)

The lungs remove excess CO2 from the blood (helping to raise the pH), and the kidneys remove excess HCO3- from the body (helping to lower the pH)

Maintains a pH of 7.4

How to generate amino acids in laboratory setting:

Methane, Ammonia, Hydrogen, and Water were added to beaker, spark was provided. A sludge material composed of amino acids was created. This experiement was designed to simulate the conditions of Earth, with spark being lightning. *Controversial still today with many scientists questioning validity

Polar Neutral Amino Acids

Serine (Ser, S) Threonine (Thr, T) Cysteine (Cys, C) Asparagine (Asp, N) Glutamine (Gln, Q) Methionine (Met, M)

Secondary Structure: a-Helix

Stabilization of secondary structure is due to H bonding. 3.6AA per turn of alpha helix Side chains protrude outwards The inside of the alpha helix appears hollow, but in reality is occupied via the different atomic radii.

Small pKa means

Strong acid

Edman Degradation

The labeled amino-terminal residue can be released without hydrolyzing the rest of the peptide. Rapidly separated by high pressure liquid chromatography

The number of pKa values differentiates polar and nonpolar amino acids from charged amino acids

The position of the pKa values for charged amino acids allows one to identify positively charged from negatively charged amino acids.

Secondary Structure: B-Sheets

The side chains are alternately above and below the plane of the strand. Parallel B-Sheet: Adjacent B strands run in the same direction. H bonds connect each amino acid on one strand with two different amino acids on the adjacent strand Anti-parallel B-Sheet: Adjacent B strands run in opposite directions. H bonds between NH and CO groups connect each amino acid to a single amino acid on an adjacent strand, stabilizing the structure. *Reference Slide 9 for figure on Structure differences.

Distribution of amino acids in Myoglobin: (Tertiary)

The surface of the molecule has many charge amino acids, as well as some hydrophobic amino acids. *Mostly HYDROPHOBIC amino acids are found on the INSIDE of the structure *Most HYDROPHILIC amino acids are found on the protein SURFACE

Comparisons between experimental and literature pKa values can allow the identification of a specific amino acid.

Titration curves are helpful in identification of AA. They will always have the same pI.

Why go from DNA to RNA first? Of note, Dr. Patel is an RNA Biologist (exams could be dense in this topic)

To prevent damage to the original DNA sequence.

Sequencing:

Use of proteases to cleave the polypeptide into shorter peptides followed by sequencing of the shorter peptides.

Acid-Base buffers confer resistance to a change in the pH of a solution when hydrogen ions (protons) or hydroxide ions are added or removed. An acid-base buffer typically consists of a _______, and its _______.

Weak acid, conjugate base (salt)

What is the technique used for identifying tertiary structure?

X-ray Crystallography

a-helix

a coiled conformation, resembling a right-handed spiral staircase, for a stretch of consecutive amino acids in which the backbone NH group of every residue donates a H-bond to the CO group of every residue.

Disulfide bridge:

a covalent bond formed when the reduced S-H groups of 2 cysteine residues react with one another to make an oxidized S-S linkage

Hydrogen Bond

a noncovalent interaction between the donor atom, which is bound to a positively polarized H atom, and the acceptor atom, which is negatively polarized. *Though not covalent, the H-bond holds the donor and acceptor atoms close together

B-turn (Reverse Hairpin turn)

a tight turn that reverses the direction of the polypeptide chain, stabilized by one or more backbone H bonds. *Changes in chain direction can also occur by loops, which are peptide chain segments with no regular conformations

Van der Waals Interaction

a weak attractive force between 2 atoms or groups of atoms, arising from the fluctuations in electron distribution around the nuclei. Van der Waals forces are stronger between electronegative atoms such as those found in hydrophobic groups

Ehlers-Danlos Syndrome

caused by a defect in the structure, production, or processing of collagen or proteins that interact with collagen *Multiple subtypes of this disease. (Reference slide 3 of lecture 2)

Phosphate buffer

consists of phosphoric acid (H3PO4) in equilibrium with dihydrogen phosphate ion (H2PO4-) and H+ *Only plays a minor role in the blood because they are found in very low concentration in blood.

Base (proton acceptor) ________ concentration of OH- in an aqueous solution

increases

Mass Spectrometry separates via

mass and charge

Electrostatic Interaction

noncovalent interaction between atoms or groups of atoms due to attraction of opposite charges

pH is a measure of (H+) in a solution

pH = -log(H+)

Henderson-Hasselbach equation

pH = pKa + log [A-]/[HA] Know log(1) = 0 *Will not need calculator for these questions!

Acidosis

pH of blood falls below 7.35

Titration curve is plotted as pH vs number of equivalents (of base or acid) per mole of sample.

pH varies upon addition of acid or base to the amino acid

pOH is a measure of (OH-) in a solution

pOH = -log(OH-)

The amino acids of a protein chain are covalently joined by amide bonds, often called ________

peptide bonds

Genetic code has 64 codons which are ________ of the 4 nucleotides UCAG taken in three's.

permutations

Peptide bonds are ...

planar *In a pair of linked amino acids, 6 atoms lie in a plane

The folded structure of a protein is directly determined by its ...

primary structure

Primary Structure:

sequence of covalent bonds in proteins. Very stable. The amino acid sequence contains all the information required for protein folding.

Quaternary Structure:

the subunit structure of a protein Ex. Hemoglobin

Hydrophillic amino acids predominately located on _____

the surface of proteins