Protein Functions: MLS 064 Biochemistry PDF

PROTEIN FUNCTIONS MLS 064: BIOCHEMISTRY PREPARED BY: NORJANAH I. SABER, RMT. VINCENT HARMON B. CABURATAN, RMT REX MOLLER Q. PALMES, RMT. LEARNING OUTCOMES LEARNING OUTCOME PROTEIN FUNCTION 1. Protein Structure and Stability: The variety of polypeptide chains in cells is influen...

PROTEIN FUNCTIONS MLS 064: BIOCHEMISTRY PREPARED BY: NORJANAH I. SABER, RMT. VINCENT HARMON B. CABURATAN, RMT REX MOLLER Q. PALMES, RMT. LEARNING OUTCOMES LEARNING OUTCOME PROTEIN FUNCTION 1. Protein Structure and Stability: The variety of polypeptide chains in cells is influenced by synthesis efficiency, folding ability, and environmental conditions like pH and temperature during purification. 2. Protein Quantification and Purification: Specific assays and fractionation techniques exploit a protein's unique structure and properties to measure and separate it from other molecules. 3. Protein Sequencing Methods: Proteins are sequenced by fragmenting polypeptides, using methods like Edman degradation or mass spectrometry to determine amino acid sequences. 4. Bioinformatics and Evolution: Protein sequence data are stored in online databases, enabling sequence comparisons to identify evolutionary relationships and trace the development of protein families. 5. Protein Evolution: Proteins evolve through gene duplication and divergence, with variation in the rate of evolution depending on the specific protein. ACTIVITIES Prequiz, Group Activity, Post Quiz and Laboratory Activities Polypeptide Diversity ❑ In general, proteins contain at least 40 residues or so; polypeptides smaller than that are simply called peptides. ❑ The largest known polypeptide chain belongs to the 35,213-residue titin, a giant (3906 kD) protein that helps arrange the repeating structures of muscle fibers. ❑ Multisubunit proteins contain several identical and/or nonidentical chains called subunits. Polypeptide Diversity ❑ The size range in which most polypeptides fall probably reflects the optimization of several biochemical processes: 1. Forty residues appears to be near the minimum for a polypeptide chain to fold into a discrete and stable shape that allows it to carry out a particular function. 2. Polypeptides with well over 1000 residues may approach the limits of efficiency of the protein synthetic machinery. The longer the polypeptide the greater the likelihood of introducing errors during transcription and translation In addition to these mild constraints on size, polypeptides are subject to more severe limitations on amino acid composition. The 20 standard amino acids do not appear with equal frequencies in proteins. For example, the most abundant amino acids in proteins are Leu, Ala, Gly, Val, Glu, and Ser; the rarest are Trp, Cys,Met,andHis. Protein Purification and Analysis ❑ The task of purifying a protein present in only trace amounts was once so arduous that many of the earliest proteins to be characterized were studied in part because they are abundant and easily isolated. ❑ Molecular cloning techniques allow almost any protein-encoding gene to be isolated from its parent organism, specifically altered (genetically engineered) if desired, and expressed at high levels in a microorganism. ❑ The first step in the isolation of a protein or other biological molecule is to get it out of the cell and into solution. ❑ Many cells require some sort of mechanical disruption to release their contents. - lysing cells use some variation of crushing or grinding followed by filtration or centrifugation to remove large, insoluble particles. ❑ If the target protein is tightly associated with a lipid membrane, a detergent or organic solvent may be used to solubilize the lipids and recover the protein Protein Purification pH, Temperature, and Other Conditions Must Be Controlled to Keep Proteins Stable. 1. pH. Biological materials are routinely dissolved in buff er solutions effective in the pH range over which the materials are stable. Failure to do so could cause their denaturation (structural disruption), if not their chemical degradation. 2. Temperature. The thermal stability of proteins varies. Although some proteins denature at low temperatures, most proteins denature at high temperatures, sometimes only a few degrees higher than their native environment. Protein purification is normally carried out at temperatures near 0°C. 3. Presence of degradative enzymes. Destroying tissues to liberate the molecule of interest also releases degradative enzymes, including proteases and nucleases. Degradative enzymes can be inhibited by adjusting the pH or temperature to values that inactivate them (provided this does not adversely affect the protein of interest) or by adding compounds that specifically block their action. Protein Purification pH, Temperature, and Other Conditions Must Be Controlled to Keep Proteins Stable. 4. Adsorption to surfaces. Many proteins are denatured by contact with the air–water interface or with glass or plastic surfaces. Hence, protein solutions are handled so as to minimize foaming and are kept relatively concentrated. 5. Long-term storage. All the factors listed above must be considered when a purified protein sample is to be kept stable. In addition, processes such as slow oxidation and microbial contamination must be prevented. Protein solutions are sometimes stored under nitrogen or argon gas (rather than under air, which contains ∼21% O2) and/or are frozen at –80°C or –196°C (the temperature of liquid nitrogen). Quantification Assays ❑ Enzymatic Reaction - a process in which an enzyme, a type of protein, acts as a catalyst to speed up a chemical reaction. ❑ Coupled Enzymatic Reaction - the product of the enzymatic reaction may be converted, by the action of another enzyme, to an easily quantified substance. ❑ Immunoassays - use antibodies, proteins produced by an animal’s immune system in response to the introduction of a foreign substance (an antigen). ❑ Radioimmunoassay (RIA) - the protein is indirectly detected by determining the degree to which it competes with a radioactively labeled standard for binding to the antibody. ❑ Enzyme-Linked Immunosorbent Assay (ELISA) Quantification Assays Quantification Assays ❑ Purification Is A Stepwise Process ❑ Proteins are purified by fractionation procedures. ❑ The idea is not necessarily to minimize the loss of the desired protein, but to eliminate selectively the other components of the mixture so that only the required substance remains. Quantification Assays ❑ Salting Out Separates Proteins by Their Solubility ❑ The solubility of a protein at low ion concentrations increases as salt is added, a phenomenon called salting in. ❑ The additional ions shield the protein’s multiple ionic charges, thereby weakening the attractive forces between individual protein molecules. ❑ However, as more salt is added, particularly with sulfate salts, the solubility of the protein again decreases. This salting out effect is primarily a result of the competition between the added salt ions and the other dissolved solutes for molecules of solvent. Quantification Assays Quantification Assays ❑ Ion Exchange Chromatography Separates ❑ In ion exchange chromatography, charged molecules bind to oppositely charged groups that are chemically linked to a matrix such as cellulose or agarose. ❑ Anions bind to cationic groups on anion exchangers, and cations bind to anionic groups on cation exchangers. ❑ Perhaps the most frequently used anion exchanger is a matrix with attached diethylaminoethyl (DEAE) groups, and the most frequently used cation exchanger is a matrix bearing carboxymethyl (CM) groups. Protein Assays ❑ Ultracentrifugation Separates Macromolecules by Mass ❑ Macromolecules in solution do not respond to the earth’s gravity by settling, because their random thermal (Brownian) motion keeps them uniformly distributed throughout the solution. Only when subjected to enormous accelerations do macromolecules begin to sediment, much like grains of sand in water. Protein Sequencing ❑ The First Step Is to Separate Subunits ❑ Each polypeptide chain has an N-terminal residue. ❑ The N-terminus of a polypeptide can be determined by several methods. The fluorescent compound 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) reacts with primary amines to yield dansylated polypeptide. ❑ The N-terminal residue can also be identified by performing the first step of Edman degradation, a procedure that liberates amino acids one at a time from the N-terminus of a polypeptide. ❑ SDS-PAGE of a protein also reveals its number of different subunits Dansyl Chloride Reaction ❑ Protein Sequencing ❑ Disulfide Bonds Between and Within Polypeptides are Cleaved ❑ Disulfide bonds can be reductively cleaved by treating them with 2- mercaptoethanol or another mercaptan (a compound that contains an —SH group): Protein Sequencing ❑ Disulfide Bonds Between and Within Polypeptides are Cleaved ❑ The resulting free sulfhydryl groups are then alkylated, usually by treatment with iodoacetate, to prevent the re-formation of disulfide bonds through oxidation by O2: Protein Sequencing ❑ The Polypeptide Chains are Cleaved ❑ Various endopeptidases (enzymes that catalyze the hydrolysis of internal peptide bonds), as opposed to exopeptidases, (which catalyze the hydrolysis of N- or C- terminal residues) can be used to fragment polypeptides. ❑ Both endopeptidases and exopeptidases (which collectively are called proteases) have side chain requirements for the residues flanking the scissile peptide bond. ❑ The digestive enzyme trypsin has the greatest specificity and is therefore the most valuable member of the arsenal of endopeptidases used to fragment polypeptides. Edman Degradation ❑ Specificities of Various Endopeptidase  Edman Degradation ❑ Edman Degradation Removes a Peptide’s N-Terminal Amino Acid Residue ❑ When peptide fragments are formed through specific cleavage reactions and have been isolated, their amino acid sequences can be determined. ❑ This can be accomplished through repeated cycles of Edman degradation. ❑ In this process (named after its inventor, Pehr Edman), phenylisothiocyanate (PITC; also known as Edman’s reagent) reacts with the N-terminal amino group of a polypeptide under mildly alkaline conditions to form a phenylthiocarbamyl (PTC) adduct. Protein Sequencing ❑ Peptides Can Be Sequenced by Mass Spectrometry ❑ Mass spectrometry has emerged as the dominant technique for characterizing and sequencing proteins. Mass spectrometry accurately measures the mass-to charge (m/z) ratio for ions in the gas phase (where m is the ion’s mass and z is its charge). ❑ Electrospray ionization (ESI), a method developed by John Fenn, a solution of a macromolecule such as a peptide is sprayed from a narrow capillary tube maintained at high voltage (∼4000 V), forming fine, highly charged droplets from which the solvent rapidly evaporates. Protein Sequencing ❑ Peptides Can Be Sequenced by Mass Spectrometry ❑ Short polypeptides (