Working with Proteins Flashcards
Common Questions About Peptides and Proteins
- What is its sequence and composition?
- What is its three-dimensional structure?
- How does it find its native fold?
- How does it achieve its biochemical role?
- How is its function regulated?
- How does it interact with other macromolecules?
- How is it related to other proteins?
- Where is it localized within the cell?
- What are its physico-chemical properties?
Proteome
The proteome is the entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions.
Unlike the genome, the proteome is not an unvarying characteristic of the cell
Forma de modificar proteinas: modificações pós-traducionais, revertíveis nas cadeias laterais
Purification
Purification of a protein is an essential step in understanding their function
1• Proteins must be released from the cell to be purified
• Cells are disrupted to form a homogenate, which is a mixture of all of the components of the cell, but no intact cells.
2 Separação por centrifugação diferencial, a força sucessivamente crescente –> sobrenadante (no fim: citoplasma) vs pellet (fração nuclear–> mitocondrial–> microsomal)
{hoje em dia também: origem das células: técnicas de engenharia genética–> sobreexpressão de proteína num e.coli –> rebentar as células (choque osmótico/…)}
Proteins can be purified based on differences in their chemical properties
Separation relies on differences in physical and chemical properties:
• charge (pintar superfície: vermelho negativa (aneónica) - azul positiva (catiónica))
• size
• affinity for a ligand
• solubility
• hydrophobicity
• thermal stability
Salting out
–> Purification according to solubility!
The solubility of proteins varies with the salt concentration.
Most proteins require some salt to dissolve in water, a process called salting in (aumenta estabilidade)
As the salt concentration is increased, different proteins will precipitate at different salt concentrations, a process called salting out
Numa mistura de proteínas, 1 solúvel, a outra não: centrifugar –> sedimentar a proteina que precipitou–> pellet (a outra, solúvel, fica no sobrenadante)
Dialysis
Dialysis is a procedure that separates proteins from solvents by taking advantage of the proteins’ larger size
The protein solution is placed in a cellophane bag (manga de celofane) with pores too small to allow the protein to diffuse, but big enough to allow the salt to equilibrate with the solution surround the dialysis bag (grande quantidade de tampão sem sal, ex água)
depois agitar, esperar ~10h –> equilíbrio
Quando é necessário diminuir a quantidade de sal:
- diluir (mas acabamos com grande volume)
- diálise
Column Chromatography
• Column chromatography allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase (tampão de eluição) to mobilize the proteins.
• Proteins vão sendo resolvidas (separadas): Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later.
Depois: detetadas e recolhidas em tubos de ensaio
- Ion Exchange chromatography
- Size exclusion chromatography
- Affinity chromatography
Separation by Charge: Ion Exchange chromatography
Protein mixture is added to column containing cation exchangers–> polymer beads with negatively charged functional groups!
=Permuta aniónica!!! (Resina carregada negativamente! ex: Carbomethyl group (ionised form) ligado a celulose/ agarose)
Proteins move through the column at rates determined by their net charge at the pH being used. Proteins muito positivas interagem fortemente com a resina, proteins with a more negative net charge move faster and elute earlier.
No fim: para libertar as positivas: competidor que faz dissociar as proteinas da resina –> gradiente do sal –> vai separando conforme a carga
(também há resinas catiónicas)
Separation by size: Size exclusion chromatography (also called gel filtration)
Protein mixture is added to column containing cross-linked polymer= Resina com microporos, que acrescentam trajetos
Large proteins cannot enter the beads and exit the column first. Small proteins can enter the beads and thus have a longer path and exit the column last.
AS PROTEÍNAS NÂO INTERAGEM COM A RESINA!
Separation by binding affinity: Affinity chromatography
Affinity chromatography takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups.
Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest. –> Resina ornamentada com ligando
Only proteins with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical to which the protein is bound through the column –> competição.
Electrophoresis: for Protein Analysis
- The electric field pulls proteins according to their charge
- The gel matrix hinders mobility of proteins according to their size and shape.
(Utilizada para visualizar a composição, não como forma de purificar grandes quantidades de proteina…)
The gel is commonly polyacrylamide, so separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE.
In this case the effect of charge is eliminated by binding a negatively charged detergent, SDS (sodium dodecyl sulfate), to all the proteins that are denatured: SDS-PAGE
- SDS micelles bind to proteins and facilitate unfolding.
- SDS gives all proteins a uniformly negative charge.
- The native shape of proteins does not matter.
- The rate of movement will only depend on size: small proteins will move faster!
Proteins can be visualized after electrophoresis
Treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself.
Each band on the gel represents a different protein (or protein subunit)
1 arrastamento= smear
Ir purificando: induzir células (“enganar” e.coli)–>aparece nova banda, da proteina!; precipitação diferencial com sal; permutas iónicas –> só sobra 1 banda –> possível analisar qualidade da purificação + medir massa do péptido
SDS-PAGE Can Be Used to Calculate the Molecular Weight of a Protein
The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight –> reta de calibrarão (com log da massa dos padrões em função da distância percorrida)
Focagem Isoelétrica
Isoelectric Focusing Can Be Used to Determine the pI of a Protein
A protein mixture is placed on a gel strip (poliacrilamida) containing an immobilized pH gradient.
When voltage is applied to the gel, each protein will migrate to its pI, the location at which it has no net charge (por isso deixa de migrar).
2D Electrophoresis
Combination of Isoelectric Focusing and SDS-PAGE !
p/ direita: decréscimo do pI
p/ baixo: decréscimo da Mr (portanto canto sup esq- proteína básica muito grande; canto inf dir- proteína acídica muito pequena)
Alterations in protein levels detected by two dimensional gel electrophoresis (ex normal colon mucosa vs colorectal tumor tissue)
