Introduction+to+Proteins+III+Part+2


 * 24 August 2006**
 * Introduction to Proteins III Part 2**
 * Dr. Kandice Williams, Ph.D.**

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=**Post-Transcriptional Modification**=


 * Covalent [|post-transcriptional modification] by conjunction of “prosthetic groups” can help to augment or inhibit protein function
 * Examples of post-transcriptional modification:
 * Phosphoserine, phosphotyrosine, phosphothreonine are the most frequent modified amino acids in protein and help modulate cellular functions.
 * Requires phosphokinase enzymes
 * Hydroxyl group addition to proline and lysine stabilizes collagen fibers
 * Lack of [|vitamin C] (ascorbate) results in **scurvy** as Vitamin C helps collagen hydroxylation process of proline and lysine
 * Vitamin C is an essential cofactor to prolyl hydroxylase enzyme
 * Acetyl group to the amino terminal helps resist degradation
 * [|Ubiquitin] tags proteins for degradation.
 * Glychosylation, methylation, sumoyolation, ubiquitination, etc. modifications are relatively new and roles are unclear

=**Collagen**=


 * More than 30% of protein in the human body is some form of [|collagen] structural protein
 * Collagen is composed of 33% [|glycine] and 20% [|proline] or [|hydroxyproline]
 * Also contain amounts of lysine, hydroxylysine, and tyrosine.
 * No tryptophan or cystiene in collagen
 * α-collagen – basic polypeptide procollagen unit with left-hand helical structure
 * Most helices are right-handed
 * β-collagen – double helix of procollagen
 * γ-collagen – triple α-helix of procollagen, formed in the ER
 * Collagen fibrils are arranged differently in various tissues:
 * Tendon – in parallel bundles
 * Skin – in sheets of fibrils layered at many angles
 * Cartilage – no distinct arrangement
 * Cornea – planar sheets stacked crossways to minimize light scatter

=**Stability of Proteins**=


 * Proteins are only marginally stable because the free energy difference between a typical folded and unfolded protein is very small
 * Marginal stability is biologically advantageous, providing flexibility for protein function
 * Enzymes undergoing conformational changes to induce fit of a specific substrate
 * 30%-40% of all proteins in the body can perform different tasks, binding to more than one molecule depending on different task requirements

=**Studying Protein Function**=
 * To understand protein function, investigators must:
 * Purify the protein of interest
 * Determine the primary amino acid sequence

**Purification of Protein**

 * Purification is necessary to determine the amino acid sequence of a specific protein
 * Determine family of proteins – protein function can be inferred from this information
 * Can compare protein between different organisms to determine evolutionary relationship
 * Similar proteins are likely to be essential to life
 * Knowledge of amino acid sequence can help determine biochemical function
 * Some amino acid sequences have been identified as signals for destination or processing of that protein, i.e. membrane or nuclear localization
 * Can develop antibodies against protein to further improve purification or allow clinical investigation
 * Amino acid sequence can be used to make DNA probes for the genetic sequence for study
 * Purification of protein can allow [|crystallography] and x-ray data to help determine the tertiary form

**Purification Steps**

 * Differential centrifugation of a homogenate of a cell to isolate different fractions of the cell
 * Isolate nuclear faction, mitochondrial faction, or microsomal faction
 * Purification can be based on solubility, size, charge, or binding affinity
 * Dialysis can separate larger molecules from smaller molecules
 * Exploits a semi-permeable membrane and allowing the concentrated solution to equilibrate with the buffer
 * Gel filtration can be used to discriminate by size
 * Larger molecules take longer to pass through beads, so different fractions can be obtained and tested for activity
 * Chromatography:
 * Affinity chromatography can separate proteins based on a specific binding affinity for a molecule using a column matrix
 * Preferred choice if antibody is available because it can provide very specific enrichment
 * Ion exchange chromatography separates proteins based on net charge at a specific pH
 * Salting out changes the salinity at which a protein becomes insoluble and precipitates out
 * [|High pressure liquid chromatography] (HPLC) – can be used for almost all column chromatography by applying pressure to achieve higher resolving power and rapid separation.
 * Check for effectiveness of purification process:
 * Check yield or total protein content in assay solution
 * [|Gel electrophoresis] separates proteins by charge, shape, or molecular mass
 * [|SDS-PAGE] – polyacrylamide gel electrophoresis
 * Contains a large negative charge and sticks to proteins
 * Allows proteins to have the same charge but different molecular weight for separation
 * Check specific activity of protein
 * Example: LDH (lactate dehydrogenase) changes lactate and NAD+ to pyruvate, NADH and H+
 * Measure the yield of its product to determine activity, in this case, NADH is easiest to measure
 * Other purification techniques:
 * Isoelectric focusing electrophoresis
 * Separation by pI (isoelectric point) of the protein, when they have no net electric charge
 * Isoelectric focusing gels are formed contained a pH gradient which the protein solution is subjected to electrophoresis without SDS
 * Two-dimensional electrophoresis
 * Combine isoelectric focusing with SDS-PAGE to obtain very high resolution separation of proteins.
 * After SDS-PAGE separation, purified protein can be cut out and subjected to mass spectrometry
 * [|Mass Spectrometry] – determine idenity of protein by mass fragment pattern

**Determination of Amino Acid Sequence**

 * Determine look at amino acid composition
 * Hydrolyze protein to break up protein to amino acids at 110°C in 6M HCL for 24 hours
 * Separate by ion-exchange chromatography
 * Use [|ninhydrin] to measure absorbance
 * [|Edman degradation]
 * Amino-terminal residue is labeled with phenyl isothiocyanate and cleaved from the protein to be identified by chromatography procedures.
 * Process repeated until complete sequence is revealed.

=**Proteomes**=
 * Goal of many researchers now is to identify and define [|proteomes]
 * Proteins expressed by the genome that work together in a functional unit
 * Defining proteome requires information encompassing each protein, function of proteins in proteome, and interactions of the protein as a **functional unit**
 * Can include information from alternatively spliced RNA, post-transcriptional modification, etc. depending on cellular environment at any given moment

=**Objectives**=