Protein Biochemistry

What techniques are used to purify proteins and analyze their purity?

• The art and science of protein purification refers to the need to develop customized strategies that exploit unique chemical and physical properties of a target protein in order to separate it from other cellular proteins with minimal loss of biochemical activity.

• Protein purification strategies usually require the use of a highly sensitive and specific biochemical assay that identifies the target protein against a background of all other cellular proteins. Specific activity refers to the ratio of target protein activity units to the total amount of protein in the sample.

• Differential centrifugation is used to fractionate proteins contained in a cell extract on the basis of the applied centrifugal force and length of centrifugation time. The resulting separation of pellet and supernatant can be used to isolate fractions enriched for proteins localized to nuclei, mitochondria, membranes, and cytosol.

• Column chromatography is a macromolecular separation technique that is essential to most protein purification strategies. Three common types of column chromatography are gel filtration chromatography, ion-exchange chromatography, and affinity chromatography.

• Gel filtration chromatography uses porous carbohydrate beads made of dextran or agarose that separate proteins on the basis of size; large proteins flow through the column faster than small proteins, with medium-sized proteins coming off the column in intermediate fractions.

• Ion-exchange columns are used to separate charged proteins on the basis of pH-dependent charge interactions with the column matrix. Anion-exchange resins are positively charged and bind negatively charged proteins, whereas cation-exchange resins are negatively charged and bind to positively charged proteins.

• A common type of affinity column is an Ni²⁺ chelating column, which is used to purify recombinant proteins that have been engineered to contain six or more histidine residues. Recombinant His-tagged proteins bind strongly to the Ni²⁺ chelating column because of high-affinity coordination between the histidine residues and the divalent Ni²⁺ metal.

• Gel electrophoresis is a protein separation technique based on the migration of proteins through an electric field connected to a solid support matrix. The two electrodes used in gel electrophoresis are the cathode, which is negatively charged and attracts positively charged proteins (cations), and the anode, which is positively charged and attracts negatively charged proteins (anions).

• SDS-PAGE is a type of gel electrophoresis in which denatured proteins carry a negative charge due to the binding of sodium dodecyl sulfate (SDS). This gives proteins a uniform charge-to-mass ratio. SDS has a hydrophobic tail that interacts with hydrophobic residues on proteins and a negatively charged sulfate group. SDS-PAGE is used to check the purity of protein preparations and identify the molecular mass of specific proteins.

• The isoelectric point (pI) of a protein is determined by the combined pKa values of all amino acids in the protein. If a protein contains more ionizable carboxyl groups than amino groups, then it will be negatively charged at pH 7 and migrate toward the positively charged anode; if a protein contains more ionizable amino groups than carboxyl groups, it will be positively charged at pH 7 and migrate toward the cathode. Proteins stop migrating in the gel strip when they reach a position where pI = pH and the protein has no net charge.

• Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separates proteins by combining two techniques: isoelectric focusing in one dimension and SDS-PAGE in the second dimension. One variation of 2-D PAGE, called two-dimensional differential in-gel electrophoresis (2-D DIGE), uses a mixed sample of fluorescently labeled proteins to identify alterations in protein abundance as a function of cell condition.

What methods are used to sequence and synthesize oligopeptides?

• Edman degradation is a protein sequencing method that uses chemical labeling and cleavage, in combination with amino acid standards, to identify N-terminal amino acids sequentially. By fragmenting a polypeptide into small peptides using differential protease digestion, it is possible to deduce the amino acid sequence of the entire protein.

• Mass spectrometry measures the mass-to-charge ratio (m/z) of molecules, which is used to deduce the molecular mass of peptide fragments through computational analyses of the spectral data to predict the identity of proteins in a biological sample.

• The three primary types of mass spectrometry are tandem mass spectrometry (MS/MS), electrospray ionization (ESI), and matrix-assisted laser desorption/ionization (MALDI).

• Solid-phase peptide synthesis is a method to generate oligopeptides by using successive rounds of covalent linkage, washing, and deblocking to add amino acids one at a time to the carboxyl-terminal amino acid attached to a resin. Solid-phase peptide synthesis begins on the carboxyl end of the peptide and builds in the direction of the amino terminus, which is the opposite direction of in vivo polypeptide synthesis.

How are the three-dimensional structures of proteins determined?

• X-ray crystallography uses a focused X-ray beam directed at a protein crystal to obtain a diffraction pattern. From analysis of the position and intensities of the diffraction spots, an electron density map of the protein crystal can be calculated. Protein models are then built to match the regions of high electron density.

• NMR spectroscopy exploits the intrinsic magnetic properties of ¹H, ¹⁵N, and ¹³C atoms to determine their relative locations in a protein using a highly concentrated solution of purified proteins. NMR instruments contain large magnetic fields that align the nuclear spins from NMR-active nuclei, which are then perturbed by short radio-frequency pulses.

• Cryo-electron microscopy (cryo-EM) uses an electron beam to generate two-dimensional (2-D) projections of three-dimensional (3-D) protein complexes. The collection of 2-D projections is computationally enhanced and used to construct a 3-D model of large multi-subunit protein complexes.

• The AlphaFold2 algorithm is an artificial intelligence–based computational tool that can be used to refine cryo-EM models on the basis of protein models contained in the DeepMind protein database. AlphaFold2 predicts 3-D structures in regions of the protein for which no X-ray or NMR structural data are available.