Biol 12: How do we analyze cells?

Non-covalent interactions

1. Ionic bonds

2. H-bonds

3. Hydrophobic interactions

Transient interactions

_______________ between molecules depend on non-covalent bonds, critical aspect of protein-protein interactions and also enzyme-substrate interactions

Methyl

-CH3

Hydroxyl

-OH

Carboxyl

-COOH

Amino

NH2

Phosphate

-PO4, a functional group in organic molecules that contributes to energy transfer, such as ATP.

Carbonyl

C=O

Sulfhydryl

-SH, a functional group that can form disulfide bonds, playing a key role in protein structure.

pH

-log[H+]

Low pH

________ will push the equilbrium toward the protonated state

High pH

________ will push the equilbrium toward the deprotonated state

Non-polar R-groups

1. Alanine

2. Valine

3. Leucine

4. Isoleucine

5. Methionine

6. Phenylalanine

7. Tryptophan

Polar non-charged R-groups

1. Serine

2. Threonine

3. Glutamine

4. Asparagine

5. Tyrosine

Polar charged R-groups

1. Aspartic acid

2. Glutamic acid

3. Lysine

4. Arginine

5. Histidine

R-groups with unique properties

1. Glycine

2. Cysteine

3. Proline

Flexibility in environment

Special property of glycine

Disulfide link

A covalent bond formed between the sulfur atoms of two cysteine residues, contributing to protein stability and structure.

Helix breaker

Proline creates kinks in polypeptide chains because of its R-group binding to the N of the amino group as well as the alpha carbon, making it regularly a __________

Primary structure

Linear sequence of AA

Secondary secondary

Local regions of structure, beta sheets and alpha helices

Tertiary structure

Overall 3D shape of a polypeptide

Quaternary structure

Assembly of subunits of 2 or more proteins together

C=O and N-H

Alpha helix is stabilized by H-bonds between _______ of different peptide bonds

protrude out

R groups ________ from helix, they are not part of the backbone

C=O and N-H

Beta sheets is stabilized by H-bonds between _______ of different peptide bonds

Parallel or anti-parallel

Beta sheets may be _________ in structure

Motifs

Regular combinations of secondary structures

Non-covalent forces

Tertiary structure is often maintained by _____________

Domains

Structural modules within a protein, usually have distinct functinos and are often conserved and exist in multiple proteins

Ions or covalent modification

Association with __________ can change protein 3D shape and regulate it

Protein phosphatase

Protein that removes phosphate group

Protein kinase

Protein adds phosphate group

Post-traditional modification

____________ can affect:

1. Interactions

2. Conformation

3. Localization

4. Intrinsic activity

Coiled-coil motif

is a structural motif in proteins that consists of two or more alpha-helices coiled around each other, often involved in protein dimerization and stability.

EF hand motif

is a helix-loop-helix structural motif found in a variety of proteins, primarily involved in calcium binding. It facilitates protein-protein interactions and signal transduction.

Immunogold TEM

combines the specificity of immunolabeling with the high resolution of electron microscopy, secondary antibody is conjugated with gold nanoparticles

Primary cell culture

is the process of isolating and maintaining cells from a living organism (usually animals), most divide for limited period of time

Cell line

is a culture of cells that can divide indefinitely in vitro, derived from a primary cell culture

Cell lysis techniques

1. Sonification

2. Force cells through small hole

3. Use detergent to make holes in the membrane

4. Tissue homogenizer (leaves organelles intact)

Density gradient equilibrium centrifugation

Technique where components will sediment in a density gradient until they reach their own buoyant density

1. Separating organelles from each other

2. Separating membranes from each other

Sodium dodecylsulfate

Less gentle detergent that solubilizes protein but also unfolds it (ionic)

Triton x-100

More gentle detergent that solubilizes protein while retaining secondary and tertiary structureand is often used in cell lysis without denaturing proteins. (non-ionic)

Detergents

Integral membrane proteins can be solubilized by using _______

amphipathic

Non-ionic detergents are ________

Types of chromatography

1. Ion-exchange

2. Gel-filtration

3. Affinity

Gel filtration chromatography

technique where smaller proteins that can fit in the pores of the gel beads spend more time in the pores and come out in later fractions than bigger proteins

Affinity chromatography

A technique that separates proteins based on their specific binding interactions with ligands attached to a stationary phase, allowing for the purification of target proteins from complex mixtures.

Co-immunoprecipiation

Affinity chromatography technique that can be used to purify protein complexes. Antibodies are on the beads and any proteins still interacting with the antigens will be purified

Aliquot

A portion of a sample that is taken for analysis or experimentation, ensuring consistency in testing and measurement.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins treated with:

• Beta-mercaptoethanol which reduces disulfide bonds

• Heat helps unfold and denature

• SDS denatures and coats proteins with negative charge stochiometrically

Reduces disulfide bonds

What does beta-mercaptoethanol do in SDS-PAGE?

Western blotting

technique used to detect specific proteins in a sample after separation by SDS-PAGE, involving transfer to a membrane and use of antibodies for visualization (immunoblotting)

Chemiluminescent detection for immunoblots

Immunoblotting technique where the secondary antibody is conjugated to an enzyme. when the substrate is incubated with membrane, light is emitted where the enzyme acts on the substrate

2D electrophoresis

A technique that separates proteins based on their isoelectric point and molecular weight

Mass spectrometry

A technique where proteins can be excised from the gel, digested with trypsin, and then analyzed by comparing the experimentally derived mass to trypsin fragments of known proteins