Proteins

Protein Isolation

• cells are collected from tissue or cell structure

• Cells are lysed on a buffered solution and sonicated, sheared or incubated in mild detergents to disrupt cell membranes

What keeps the protein inactive and active during purification?

• protease inhibitors - freeze protease enzymes from chopping up proteins

• reducing agents - prevent oxidation of disulfide bonds

• cold temps - slows down enzymatic activity

To purify a protein you must:

• break down the cell tissue via suspension(using enzymes)

• lyse the cells via sonification, shearing, or mild detergents

• cell is them homogenized

Gel Filtration Chromatography

• Size Exclusion Chromatography.

• Separated based on molecular size.

• Large proteins elute first

Ion Exchange Chromatography

• Separated based on net surface charge

  • cation exchange: negative beads bind cationic proteins(net positive charge)

  • anion exchange: positive beads bind anionic proteins (net negative charge)

• You can elute by raising salt concentrations

Affinity chromatography

• Separation based on specific interactions between a protein and a ligand.

• Non binding proteins elute first

• To elute, add free competing lignin to make the protein let go and bind to new ligand

Native Gels vs Denaturing Cells

Native cells separate by size and charge

Denaturing Gels (SDS-PAGE) separate primarily by size

Polyacrylamide and Agrose gels

• Types of gels used for electrophoresis, with polyacrylamide commonly used for proteins and agarose for larger DNA molecules. Polyacrylamide offers high resolution for small molecules, while agarose provides better separation for larger fragments.

Gel Electrophoresis

• electric current applied to gel to pull molecules through the system

• molecules visualized by staining

• small molecules move faster

• cells move from negative charge to positive charge

SDS-Polyacryamide Gel Electrophoresis (PAGE)

• Proteins boiled on solution of SDS to denature them and give negative charge

• Reducing agent added to break disulfide bonds

• Most accurate for size estimate

• Protein shape is not a factor

2-Dimensional Gel Electrophoresis

• proteins separated according to charge as a function of pH by isoelectric focusing

• Loaded into SDS-PAGE to be separated by mass

X-Ray Crystallography

• X-ray beam aimed at protein crystal and protein crystals will deffract

• Diffraction patterns captured by x-ray detector and data is interpreted by software

• Limitations: growing crystals and structure determination are both difficult

NMR Spectroscopy

• reveals relative locations of atoms by leveraging nucleus magnetic properties

• provides solution-state structures and is uniquely suited to studying dynamics and folding states

• Limitations: Requires large amounts of pure sample and is less effective for very large proteins.

What are the most common nuclei used to study protein dynamics

1H, 15N, and 13C

AlphaFold

An AI-based system developed by DeepMind to predict protein structures from amino acid sequences with high accuracy.

• AlphaFold DB: >200million predicted structures

• Per-residue confidence score (pLDDT) flags reliable vs uncertain regions

What is a caveat to the AlphaFold

it may not always predict the dynamics or interactions of proteins accurately, as it primarily focuses on static structures.

Proteome

The entire set of proteins expressed by an organism, cell, or tissue at a specific time and under specific conditions.

Transcriptome

The complete set of RNA transcripts produced by the genome at any given time, reflecting gene expression in a specific cell or condition.

Why does the proteome give an accurate reflection of what the cell is doing at any given moment?

Bc proteins are agents of cellular function