Altius: Biochemistry 1
Electrophoresis for proteins
- separates proteins based on size 1. Proteins are denatured and coated with uniform negative charge 2. they are ran across the gel and the smaller proteins will travel faster and farther
Protein folding
- translated proteins assume secondary structure instantly after being made and then later into tertiary state which is driven by multiple interactions
Trypsin
an enzyme from the pancreas that digests proteins in the small intestine - targets positive R group of lysine and arginine
What are 'residues'?
another name for amino acid
Provide examples of Immune Proteins:
antigens and antibodies
If the side chain contains an amine functional group, the amino acid produces a:
basic solution because the extra amine group is not neutralized by the acid group
Oxidoreductases function:
catalyze oxidation-reduction reactions NAD+ = oxidizing agent (removes Hydrogen) NADH = reducing agent (adds Hydrogen)
Molten globule (folding state):
partially folded
Enzymes are sensitive to what?
ph, temp
What are the two ways to separate proteins?
via isoelectric point (isoelectric focusing) Electrophoresis
Amino acids with basic side chains (characteristics)
Often involved at the active sites of enzymes, in hydrogen bonding interactions and in acid/base type reactivity (e.g. histidine)
Chymotrypsin
One of the main pancreatic proteases; - breaks down phenylalanine, tryptophan, and tyrosine
Amino acids with acidic side chains (characteristics)
These carboxylate group will be -ve at physiological pH. Often involved at the active sites of enzymes, in hydrogen bonding interactions and in acid/base type reactivity.
pI of a neutral amino acid
(pKa of NH2 group + pKA of -COOH group) / 2 average pka of COOH and NH2
pI of an acidic amino acid
(pKa of R group + pKa of -COOH group) / 2 average of pKa-acidic R group and COOH group
pI of a basic amino acid
(pKa, NH3+ group + pKa, R group)/2 average pKa NH3+ and pKa-basic R group
Are natural amino acids L or D and describe position of amine group on a fisher projection?
- All natural and native amino acids are levorotatory (L) In a Fischer projection, (L) configuration results in Amine on left side (D) configuration results in amine on right side *COOH group must be on top*
alpha carbon of amino acid
- holds all the substituents - Glycine is the only achiral amino acid with 2 hydrogens on alpha carbon
What do catalysts and enzymes have in common
-both lower activation energy - increase rxn rate - do NOT affect equilibrium - do NOT affect K(eq) - do NOT affect yield or percent yield
Acid-Base Functionality of Amino Acids:
Amino acids = WEAK acids ▪ Each amino acid has a MINIMUM of two acidic protons: -COOH and -NH3+ ▪ Some amino acids (discussed below) have acidic side chains, and therefore three acidic protons. ▪ Per the above statements, each amino acid has either two or three pKa values. ▪ The amino acid acts as a buffer when the pH is near the pKa of one of the acidic protons
Order of deprotonation in an amino acid (and associated pKa's):
1. alpha -COOH Group (pKa ~ 2) 2. -R Group = ACIDIC (pKa ~ 4) [Asp = 3.7 ; Glu = 4.5] 3. -R Group, His (pKa ~ 6) 4. alpha -NH3 + Group (pKa ~ 9) 5. -R Group = BASIC (pKa ~ 11-12) [Lys = 10.7; Arg = 12]
How many acidic protons at MINIMUM does each amino acid have?
2 - COOH - NH3+
oligopeptide
4-10 amino acids or just a small chain of amino acids
Peptides are Written, Read, AND Synthesized from:
N-terminus → C-terminus
describe interactions involved with: Solvation Layer
A layer of water that surrounds a dissolved protein. The water molecules in this layer interact closely with each other and with the protein's surface. The water in the hydration layer is more ordered that the bulk water in the general area and is considered not to participate with the bulk (a.k.a., unstructured) water when considering colligative properties.
Strecker Synthesis of Amino Acids
A method of synthesizing amino acids that uses condensation between an aldehyde and hydrogen cyanide, followed by hydrolysis.
isoelectric focusing
A specialized method of separating proteins by their isoelectric point using electrophoresis; the gel is modified to possess a pH gradient - separates by isoelectric point - more basic will be attracted toward negative end - more acidic will be attracted toward positive end
Basic structure of amino acid
Alpha (central) carbon, R group (functional side chain), amino and carboxyl group, and hydrogen
Polarity Ranking of the Functional Groups: (most polar first)
Amide > Acid > Alcohol > Ketone ~ Aldehyde > Amine > Ester > Ether > Alkane
Solubility of amino acids:
Amino acids are generally soluble in water and insoluble in non-polar organic solvents such as hydrocarbons. This again reflects the presence of the zwitterions. In water, the ionic attractions between the ions in the solid amino acid are replaced by strong attractions between polar water molecules and the zwitterions. This is much the same as any other ionic substance dissolving in water. The extent of the solubility in water varies depending on the size and nature of the "R" group.
Gabriel Synthesis - amino acid synthesis
An amino acid is generated from phthalimide (nucleophile) and diethyl bromomalonate, using two SN2 reactions, hydrolysis, and decarboxylation.
describe interactions involved with: Proline Turns
Can be considered as either disrupting 2° structure or as contributing to 3° structure. Neither alpha helices, nor beta sheets can contain proline internally without disruption of the 2° structure. However, proline residues are often found at the beginning of alpha-helices and are very common (along with glycine) in the sharp turns at the end of two adjacent rows in a beta-sheet.
What type of reaction is peptide formation?
Dehydration synthesis and Acyl substitution The amine group nitrogen (nucleophile) from the NEW amino acid attacks the carbonyl carbon (electrophile) on the C-TERMINUS of the growing peptide chain (aided by the enzymatic function of the ribosome)
Essential vs. non-essential amino acids
Essential amino acids are not produced in our body so they are essential to our diets.
describe interactions involved with: Entropy and Protein Folding
Even when water interacts with a dissolved polar solute, this interaction is less entropically favorable than those same water molecules interacting with only other water molecules. However, the driving thermodynamic force that favors protein folding results from the fact that non-polar regions require a much GREATER ordering of water molecules to accomplish solvation. Therefore, transitioning from solvation of non-polar regions to solvation of a mostly polar or charged globular protein surface, represents a net increase in entropy. In fact, it is enough to overcome the decreased entropy associated with the protein being in a folded rather than an unfolded state. This favorable increase in entropy is a major contributor to the overall conformational stability of the folded protein.
describe interactions involved with: Salt Bridges
Formed when acidic and basic -R groups undergo a neutralization reaction resulting in a salt.
tertiary structure
Geometric 3D folding of alpha helices and beta sheets to form a functional or globular protein Interactions of tertiary structure: 1. H Bonding 2. Disulfide bridge - cysteine 3. Hydrophobic/hydrophilic interactions 4. Ionic character: charge-charge interactions between + and - amino acid 5. Vander wall forces - wtf 6. proline turns - kinky ;)
Most amino acids are L and S EXCEPT?
Glycine (nonchiral) and cysteine (has high ranking sulfur group)
describe interactions involved with: Hydrogen Bonds
Hydrogen bonding between -R groups also encourages folding and stabilizes the folded protein.
Describe the structure and bonding interactions of Beta Sheets:
Hydrogen bonding between ALL of the carbonyl oxygens in one row and the amide hydrogens in the adjacent row. • ALL residues involved in hydrogen bonding! • R groups are directed perpendicular to the plane of the beta sheet, on both sides. • Beta sheets assume a pleated conformation. This is necessary for the carboxyl and amide moieties to line up properly so that every residue is participating in two hydrogen bonds.
describe bonding and interactions in Alpha Helices:
Hydrogen bonding between the carbonyl oxygens and the amide hydrogens that are exactly FOUR residues apart, including the residues involved in the hydrogen bond (i.e., A-B-B-A arrangement where A and A share a hydrogen bond) Each amino acid forms a hydrogen bond with the fourth amino following it in the chain.
describe interactions involved with: Hydrophobic Core
Hydrophobic -R groups fold into the interior of a globular protein to escape water. They often bring some smaller polar groups with them, which interact in a complementary way to stabilize the folded protein further.
What determines protein folding?
Hydrophobic R groups fold inwards (into the protein core) Hydrophilic R groups fold outward on the surface of the protein Proteins with low hydrophobicity do not fold into a stable structure, but can retain function (e.g., Intrinsically Disordered Proteins)
What determines if a substrate will bind to a potential active site?
If complementary charges and hydrophilicity and hydrophobicity agree (on -R groups)
Why do amino acids have such high melting points?
Instead of the weaker hydrogen bonds and other intermolecular forces that you might have expected, you actually have much stronger ionic attractions between one ion and its neighbours. These ionic attractions take more energy to break and so the amino acids have high melting points for the size of the molecules.
describe interactions involved with: Electrostatic Interactions
Interactions between charged -R groups both encourage the act of folding itself, and stabilize the protein in its folded state.
What steps are necessary to cause a simple denatured protein to re-fold? Will this process work, unaided, for all proteins?
It is often not possible to re-fold unfolded proteins. In cases where you can, proteins have been slowly denatured using a mild denaturant, something that will break hydrogen bonds and disulfide bonds but not other covalent bonds. When denaturing a protein that you want to re-fold, you want to denature slowly. Too quickly or too harshly and irreversible denaturing will occur, such as collapse of all hydrophobic amino acids into the center of the protein. To refold a denatured protein, you want to slowly remove the denaturant from solution. As you slowly wash away the denaturant, secondary structure will start to return, followed by tertiary and quaternary. This will not work with proteins that have been heat denatured, as this often disrupts hydrophobic interactions to the point that all hydrophobic residues collapse together. This will create hydrophobic interactions so strong that it cannot be unfolded. Other types of denaturation that are difficult or impossible to reverse: proteins that are subject to extremes in pH or harsh denaturants that may dissolve covalent bonds. If the first step of the reaction involves protonating the substrate, the active site amino acids will need to hydrogen bond with the atom gaining the proton. In this case, the amino acids will be accepting the hydrogen in the hydrogen bond (since the substrate is gaining its proton). Likely amino acids are aspartic acid and glutamic acid, since the carboxylic acid groups can readily bond with the new proton.
positive cooperativity
Ligand affinity increases with the binding of each subsequent ligand. In the case of hemoglobin, affinity for the first oxygen is relatively low, but increases for the second, third, and fourth oxygen to bind. This affinity remains in effect during offloading of oxygen at the tissues. Therefore, the first oxygen (highest cooperative affinity) dissociates at the slowest rate, but each subsequent oxygen is released more easily.
How are pKa and pH related?
Pka of a functional group or a molecule will tell how acidic or not acidic it is pH is a property of a particular solution that tells us the concentration of protons or hydroxides
If enzyme pocket is negative and polar, What will substate binding spots be?
Positive and polar positive attracts negative BUT, Polar loves polar Nonpolar loves non polar
What determines reaction mechanisms in enzyme pocket?
R group chemistry of the amino acids in contact with substrate
What is the positioning of R groups in Alpha Helices?
R groups are directed exactly away from the alpha helix cylinder (i.e., perpendicular to a plane tangent to the surface of the alpha helix).
How does resonance affect amino acid structure?
Resonance between the pi electrons of the C=O bond and the nitrogen lone pair of the C-N bond yields two resonance structures for any peptide bond. The actual structure is a hybrid of the two, and therefore: BOTH the C=O bond and the C-N bond in a peptide bond have DOUBLE BOND character. Double bond character → RIGID peptide bond with limited rotation.
What happens when pH is lower than pKa value of a functional group?
That function group will get protonated and could possibly obtain a positive charge
Acyl Substitution of amino acids
The amine group (NUC) on new amino acid attacks carbonyl carbon (electrophile) on C-Terminus
Amino acids with non-polar side chains (characteristics)
The hydrophobic side chains are chemically unreactive and tend to aggregate rather than be exposed to the aqueous environment, so they tend to found on the interior of proteins. Hydrophobic means "water hating" - remember "oil and water don't mix" and "like dissolves like" - this is because non-polar hydrocarbons do not interact with polar water molecules in an energetically favourable way - they would rather interact with another non-polar hydrocarbon molecule : this it the hydrophobic effect - the aggregation of non-polar systems in an aqueous environment.
isoelectric point
The isoelectric point (pI) is the pH at which a molecule carries no net charge. For amino acids and other organic molecules, the molecule is often in the form of a zwitterion. For molecules that have two pKas, the equation for pI is: pI = (pKa1 + pKa2)/2. At pH lower than the pI, the molecule will have a net positive charge. At a pH higher than the pI, the molecule will have a net negative charge. This can be utilized to separate molecules based on their pIs by varying the pH in a gel. The isoelectric point is most similar to the equivalence point in acid-base titration.
describe interactions involved with: Hydrophilic Surface
The majority of the -R groups on the surface of a globular protein are either polar or charged.
What happens when the pH is ABOVE the pKa of a given group?
The molecule will get deprotonated
What aspect of a protein is primarily responsible the manner in which it folds?
The primary sequence of a protein is primarily responsible for how it folds. There is evidence for this in the fact that simple proteins fold spontaneously without chaperone proteins. Also, as peptides are exiting ribosomes, they immediately begin folding. This makes sense if you think about it: certain amino acids are found in certain secondary structures, and some groups of amino acids found together will almost always fold in the same way. Mutations in key amino acids can completely destroy secondary and tertiary structure, while other mutations may alter only segments of folding.
Amino acids with polar side chains (characteristics)
These are side chains can be involved in hydrogen bonding interactions. Cysteine is important because of its ability to form disulfide bonds.
Transferases function:
Transfer of a functional group (e.g., kinases, aminotransferases) Peptidyl transferase is an example which moves amino acid from tRNA and adds it to growing peptide chain
Protein Hydrolysis: (provide 2 examples of proteases and the specific amino acids they cleave)
Trypsin and chymotrypsin cleave proteins on the CARBOXYL SIDE of specific amino acid residues: • Trypsin = arginine, lysine • Chymotrypsin = phenylalanine, tryptophan, tyrosine
describe interactions involved with: Disulfide Bonds
Two oxidized cysteine residues form a disulfide (R-S-S-R) bond. This is the strongest type of protein folding interaction. Disulfide bonds between keratin alpha helices are what make hair more or less curly
What is typically observed about Proline in secondary structures?
Usually the first residue at the very end of an alpha helix, but rarely found inside the helix because it introduces a KINK/TURN. This same KINK/TURN is desirable at the end of beta-sheets because the chain must make a 180 degree turn to align as a neighboring row in the beta sheet. because of proline's unusual cyclical shape, introducing a proline into an alpha helix or beta sheet will cause a kink. Proline turns are also found at the end of most strands involves in beta sheets. The sharp turn helps the chain redirect in such a way that the next segment is running antiparallel to the previous segment in the sheet formation
When you see PROTEIN or ENZYME THINK:
What amino acids are present and what is the chemistry of their -R groups?
Sulfur Linkage
When 2 cysteine molecules bind
Stabilizing features of amino acids with a substrate:
You are only looking for COMPLENTARITY. Generally speaking, opposite charges will attract and stabilize each other, polar molecules will be attracted to each other and stabilize one another, non-polar moieties will aggregate away from water stabilizing the molecules involves, and any functional group capable of hydrogen bonding with another functional group will form a strong stabilizing association. For H-bond associations remember to consider whether the functional group can act as an H-bond donor, an H-bond recipient, or both. More specifically, for this question, think about the types of interactions that will stabilize something charged, polar, or non-polar
peptide bond is:
a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O). This is a dehydration synthesis reaction (also known as a condensation reaction), and usually occurs between amino acids This linkage, called a peptide bond, has several important properties. First, it is resistant to hydrolysis so that proteins are remarkably stable kinetically. Second, the peptide group is planar because the C-N bond has considerable double-bond character. Third, each peptide bond has both a hydrogen-bond donor (the NH group) and a hydrogen-bond acceptor (the CO group). Hydrogen bonding between these backbone groups is a distinctive feature of protein structure. Finally, the peptide bond is uncharged, which allows proteins to form tightly packed globular structures having significant amounts of the backbone buried within the protein interior. Because they are linear polymers, proteins can be described as sequences of amino acids. Such sequences are written from the amino to the carboxyl terminus.
-R groups (in amino acids) with negative charges are ____________ and with positive charges are ____________
acidic ; basic
Provide examples of Structural Proteins:
actin (thin filaments, microfilaments) tubulin (microtubules) keratin (hair and nails, intermediate filaments) elastin (connective tissue, extracellular matrix)
-R group function of amino acids:
determine chemistry of amino acid and folding pattern
Amino acids with an amide on the side chain:
do not produce basic solutions
Protease
enzyme that breaks down proteins
Globule (folding state):
fully folded
Molten (folding state):
fully unfolded; aka denatured
Provide examples of Binding Proteins:
hemoglobin, calmodulin, troponin, tropomyosin, histones, transcription factors, cell adhesion molecules
Hydrolase function:
hydrolysis
secondary structure of protein:
local 3D configuration of peptide chain ALPHA HELIX/ BETA SHEETS are secondary structure
Describe the movement of Dynein:
move on microtubules from (+) to (-) periphery to center of the cell nerve cell dendrite → cell body
Describe the movement of Kinesin:
move on microtubules from (-) to (+) center to periphery of cell nerve cell body → dendrite
quaternary structure
multiple folded proteins into a multi subunit complex ex: hemoglobin
Provide examples of Motor proteins:
myosin (power stroke, cellular transport) kinesins and dyneins (vesicles, cellular transport, cell division, cilia, flagella)
Isomerase function:
rearrange the existing atoms of a molecule, that is, create isomers of the starting material
primary protein structure
sequence of amino acids
List all amino acids that could be logical "stabilizing features" of the enzyme pocket for an enzyme whose substrate is: non polar
stabilized by alanine, glycine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan, via hydrophobic interactions
List all amino acids that could be logical "stabilizing features" of the enzyme pocket for an enzyme whose substrate is: polar
stabilized by asparagine, glutamine, aspartic acid, glutamic acid, lysine, and arginine, via dipole interactions and via hydrogen bonding with any amines or acid groups
List all amino acids that could be logical "stabilizing features" of the enzyme pocket for an enzyme whose substrate is: positively charged
stabilized by aspartic acid, glutamate, asparagine, and glutamine, via positive-negative charge interactions, and via hydrogen bonding with the carboxyl group of asparagine and glutamine
List all amino acids that could be logical "stabilizing features" of the enzyme pocket for an enzyme whose substrate is: negatively charged
stabilized by lysine, arginine, serine, threonine, tyrosine, and asparagine, via positive-negative charge interactions, and via hydrogen bonding with the alcohol group of serine, and threonine, or with the amine group of tyrosine and asparagine
The primary structure refers to:
the amino acid sequence
The secondary structure refers to:
the conformation adopted by local regions of the polypeptide chain Two major elements of secondary structure are the α helix and the β strand. In the a helix, the polypeptide chain twists into a tightly packed rod. Within the helix, the CO group of each amino acid is hydrogen bonded to the NH group of the amino acid four residues along the polypeptide chain. In the β strand, the polypeptide chain is nearly fully extended. Two or more β strands connected by NH-to-CO hydrogen bonds come together to form β sheets.
N-terminus
the end of a polypeptide or protein that has a free amino group
C-terminus
the end of a polypeptide or protein that has a free carboxyl group
The number of alkyl groups also influences the polarity. The more alkyl groups present:
the more non-polar the amino acid will be
Tertiary structure describes:
the overall folding of the polypeptide chain The compact, asymmetric structure that individual polypeptides attain is called tertiary structure. The tertiary structures of water-soluble proteins have features in common: (1) an interior formed of amino acids with hydrophobic side chains and (2) a surface formed largely of hydrophilic amino acids that interact with the aqueous environment. The driving force for the formation of the tertiary structure of water-soluble proteins is the hydrophobic interactions between the interior residues
quaternary structure refers to:
the specific association of multiple polypeptide chains to form multisubunit complexes. Proteins consisting of more than one polypeptide chain display quaternary structure, and each individual polypeptide chain is called a subunit. Quaternary structure can be as simple as two identical subunits or as complex as dozens of different subunits. In most cases, the subunits are held together by noncovalent bonds.
Amino Acid Interactions in an Enzyme
• Hydrophobic core • Hydrophilic surface • Electrostatic interactions- between charged parts • Hydrogen bonds- encourages folding • Disulfide bond: STRONGEST • Salt bridges: formed when an acidic and basic R group undergo a neutralization reaction • Solvation layer: interaction when protein is dissolved in water
Provide two common examples of secondary structures in real life:
• Keratin, found in hair and nails = alpha helices. • Fibroin, the molecule that makes up silk = beta sheets.
protein denaturing agents
▪ Acid ▪ Heat ▪ Urea ▪ Mercaptoethanol