Proteins and Their Purification

These flashcards cover key concepts from the lecture notes on proteins and their purification methods, helping to reinforce the main ideas for exam preparation.

What are the basic components that all amino acids share in their structure?

All amino acids (except proline) share an \alpha-carbon, an amino group (\text{NH}_3^+), a carboxyl group (\text{COO}^-), hydrogen, and a side chain (R group).

At physiological pH, amino acids exist as __.

Dipolar ions (zwitterions) with an ammonium group (\text{NH}_3^+) and a carboxylate group (\text{COO}^-).

Which common amino acid is achiral?

Glycine, as its R-group is simply a hydrogen atom.

What configuration do most naturally occurring amino acids found in proteins have?

L-configuration at the \alpha-carbon.

What are the approximate pKa values for the \alpha-carboxyl group and \alpha-amino group in free amino acids?

\alpha-carboxyl group: 2.0 - 2.5; \alpha-amino group: 9.0 - 10.0.

What is the significance of the isoelectric point (pI)?

The pH at which the net charge of a protein is zero.

How do you calculate the pI for neutral amino acids?

pI = (pKa1 + pKa2) / 2.

What distinguishes nonpolar (hydrophobic) amino acids?

They tend to be buried inside proteins, avoiding water interactions.

What defines an amino acid as nonpolar (hydrophobic)?

It has R-groups composed primarily of hydrocarbons, which tend to avoid water.

List the nonpolar, aliphatic amino acids.

Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, and Proline.

What distinguishes nonpolar, aromatic amino acids?

Phenylalanine (very nonpolar), Tyrosine (has -OH, slightly polar), Tryptophan (has indole ring, slightly polar).

What defines an amino acid as polar, uncharged?

It has R-groups with functional groups capable of hydrogen bonding (e.g., -OH, -SH, -CONH_2), but no net charge at physiological pH.

List the polar, uncharged amino acids.

Serine, Threonine, Cysteine, Asparagine, Glutamine.

Which amino acid can form disulfide bonds?

Cysteine, through oxidation of its sulfhydryl (-SH) group.

List the acidic amino acids and their charged forms at physiological pH.

Aspartate and Glutamate; their side chains contain carboxyl groups which are deprotonated (\text{COO}^-) and negatively charged.

List the basic amino acids and their charged forms at physiological pH.

Lysine, Arginine, and Histidine; their side chains contain amino or guanidinium groups that are protonated and positively charged.

Which basic amino acid has a side chain pKa near physiological pH, allowing it to act as a buffer?

Histidine, with an imidazole ring pKa of approximately 6.0.

What occurs during peptide bond formation?

A condensation reaction occurs between the \alpha-COOH of one amino acid and the \alpha -NH_2 of another, producing water.

What are the key characteristics of a peptide bond?

It has partial double-bond character (due to resonance), making it rigid, planar, and unable to rotate freely.

What is the N-terminus of a polypeptide chain?

The end with a free \alpha-amino group.

What is the C-terminus of a polypeptide chain?

The end with a free \alpha-carboxyl group.

What defines the primary structure of a protein?

The specific linear sequence of amino acids linked by peptide bonds.

What is the main force stabilizing secondary structures like \alpha-helices and \beta-sheets?

Hydrogen bonds between the backbone carbonyl oxygen and amide hydrogen atoms.

Describe an \alpha-helix.

A rigid, rod-like coiled structure where the polypeptide backbone winds around a central axis, stabilized by H-bonds between C=O of residue i and N-H of residue i+4.

Which amino acids are good \alpha-helix formers?

Alanine, Leucine, Methionine, Glutamate, Lysine (small, uncharged, or with side chains that don't clash).

Which amino acids are characterized as helix breakers?

Proline and bulky hydrophobic clusters.

Describe a \beta-sheet.

An extended, pleated structure formed by two or more polypeptide strands (\beta-strands) running parallel or antiparallel, stabilized by inter-strand backbone hydrogen bonds.

What are the two main types of \beta-sheets?

Parallel and antiparallel, differing in the directionality of adjacent strands.

What are \beta-turns (or reverse turns) and where are they typically found?

Short, abrupt turns that reverse the direction of the polypeptide chain, often found on the protein surface connecting \beta-strands or \alpha-helices.

What amino acids are frequently found in \beta-turns?

Proline (introduces a kink) and Glycine (small and flexible).

What are supersecondary structures or motifs?

Combinations of two or more secondary structures that form a recognizable folding pattern (e.g., \beta -\alpha -\beta loop, helix-loop-helix).

What is the tertiary structure of a protein?

The overall three-dimensional shape of a single polypeptide chain, including the arrangement of all its atoms.

What are the driving forces for tertiary structure formation in proteins?

Hydrophobic effects, hydrogen bonds, salt bridges, van der Waals forces, and disulfide bonds.

What structural feature is most important for the stability of proteins?

The hydrophobic effect.

Where are hydrophobic amino acid residues typically located in water-soluble globular proteins?

Buried in the interior of the protein, away from the aqueous environment.

What are protein domains?

Independently folded, compact functional or structural units within a larger polypeptide chain, often associated with a specific function.

What is the quaternary structure of a protein?

The arrangement and interaction of multiple polypeptide subunits (monomers) to form a functional multi-subunit protein complex.

What types of interactions stabilize quaternary structure?

Primarily non-covalent interactions (hydrophobic interactions, hydrogen bonds, salt bridges) and sometimes covalent disulfide bonds.

What is a key role of chaperones in protein folding?

They assist in proper folding and prevent aggregation.

What is protein denaturation?

The loss of a protein's native three-dimensional structure due to disruption of its non-covalent interactions, leading to loss of biological activity.

Name common denaturing agents.

Heat, extreme pH, organic solvents, detergents (SDS), heavy metals, chaotropic agents (urea, guanidinium chloride), reducing agents (for disulfide bonds).

What is Levinthal's Paradox?

The observation that proteins fold relatively quickly to their native state despite the enormous number of possible conformations, suggesting a guided, rather than random, folding pathway.

How is protein folding often conceptualized in terms of energy?

Using a 'folding funnel' model, where a protein explores an energy landscape, with the native state representing the global energy minimum.

What are chaperonins?

A class of chaperones (e.g., GroEL/GroES) that form large, barrel-shaped complexes to provide an isolated environment for protein folding.

What are some diseases associated with protein misfolding and aggregation?

Alzheimer's disease, Parkinson's disease, Huntington's disease, Type 2 diabetes, Creutzfeldt-Jakob disease (prion diseases).

What is the difference between myoglobin and hemoglobin in terms of structure and function?

Myoglobin has one subunit and stores O2, while hemoglobin is a tetramer that binds O2 cooperatively.

Which molecule is a monomer primarily responsible for oxygen storage in muscle tissue?

Myoglobin.

Describe the cooperativity of oxygen binding in hemoglobin.

The binding of one oxygen molecule to a heme site in hemoglobin increases the affinity of the remaining heme sites for oxygen, resulting in a sigmoidal binding curve.

What is the T (tense) state of hemoglobin?

The low-affinity deoxygenated state, stabilized by interactions between subunits.

What is the R (relaxed) state of hemoglobin?

The high-affinity oxygenated state, with fewer interactions between subunits.

What is the significance of the Bohr effect in hemoglobin function?

Lower pH increases H^+ concentration, shifting the O_2 binding curve to release oxygen.

How does carbon dioxide (CO_2) influence hemoglobin's oxygen affinity, apart from its effect on pH?

CO_2 can bind directly to the N-terminal amino groups of hemoglobin, forming carbamate, which stabilizes the T-state and reduces oxygen affinity (the carbamino effect).

How does 2,3-bisphosphoglycerate (2,3-BPG) affect hemoglobin's oxygen affinity?

2,3-BPG binds to the central cavity of deoxyhemoglobin, stabilizing the T-state and thereby decreasing hemoglobin's oxygen affinity, promoting oxygen release to tissues.

What is the primary objective of protein purification?

To isolate a specific protein from a complex mixture, usually aiming for high purity and recovery of biological activity.

What is total protein in purification metrics?

The total amount of protein in milligrams (mg) present in a given fraction.

What does total activity refer to in protein purification metrics?

The functional activity of the enzyme measured in units.

How is specific activity calculated and why is it important in protein purification?

Specific activity = Total activity / Total protein (Units/mg). It measures the purity of the enzyme, increasing with each purification step.

How does salting out (e.g., ammonium sulfate precipitation) work?

High salt concentrations reduce the solubility of proteins (by competing for water molecules), causing them to precipitate based on their distinct solubility limits.

What method is used for concentrating proteins through hydrophobic interactions?

Ammonium sulfate precipitation.

What is the principle behind ion-exchange chromatography?

Separates proteins based on their net charge, using a charged stationary phase to which proteins bind and are then eluted by changes in salt concentration or pH.

What is the principle behind affinity chromatography?

Utilizes the specific, reversible binding affinity between a protein and a ligand (e.g., an antibody, enzyme substrate analog) immobilized on a matrix for highly selective purification.

What is the principle behind size exclusion chromatography (gel filtration)?

Large proteins elute first because they cannot enter the pores of the stationary phase.

What is polyacrylamide gel electrophoresis (PAGE)?

A method to separate proteins based on their charge, size, and shape by migration through a polyacrylamide gel under an electric field.

What does SDS-PAGE do to proteins and why is it useful?

SDS denatures proteins and imparts a uniform negative charge, allowing separation based on size.

How does a protein's migration rate in SDS-PAGE relate to its molecular weight?

Smaller proteins migrate faster and further through the gel than larger proteins, allowing for molecular weight determination.

What is isoelectric focusing (IEF)?

A technique that separates proteins based on their isoelectric point (pI). Proteins migrate through a pH gradient until they reach the pH at which their net charge is zero.

What is two-dimensional (2D) gel electrophoresis?

A high-resolution separation technique that combines isoelectric focusing (separation by pI) in the first dimension and SDS-PAGE (separation by size) in the second dimension.

What is Western blotting (immunoblotting) used for?

To detect specific proteins in a sample after electrophoretic separation, using antibodies specific to the target protein.

What is mass spectrometry used for in proteomics?

To determine the accurate molecular mass of proteins and