Biochemistry Lecture Notes PDF

18 Ibrahim Aldarbi Roaa Maa’dat Nabil Bashir Properties of Proteins: Denaturation and Renaturation Denaturation • Denaturation is the disruption of the native conformation of a protein via breaking the noncovalent bonds that determine the structure of a protein. • Native means that this protei...

18 Ibrahim Aldarbi Roaa Maa’dat Nabil Bashir Properties of Proteins: Denaturation and Renaturation Denaturation • Denaturation is the disruption of the native conformation of a protein via breaking the noncovalent bonds that determine the structure of a protein. • Native means that this protein is folded, has its proper tertiary structure, and it’s functional. • Complete disruption of tertiary structure is achieved by reduction of the disulfide bonds in a protein • The denatured protein loses its properties such as activity, become insoluble and unfolded. Disulfide bonds Urea will break hydrogen bonds. Mercapto-ethanol will break disulfide bonds. Denaturing agents • Heat disrupts low-energy van der Waals forces in proteins • Extremes of pH: change in the charge of the protein’s amino acid side chains (electrostatic and hydrogen bonds). • Detergents: Triton X-100 (nonionic, uncharged) and sodium dodecyl sulfate (SDS, anionic, charged) disrupt the hydrophobic forces. • SDS also disrupt electrostatic interactions. • Urea and guanidine hydrochloride disrupt hydrogen bonding and hydrophobic interactions. • Reducing agents such as β-mercaptoethanol (β-ME) and dithiothreitol (DTT). • Both reduce disulfide bonds. Renaturation • Renaturation is the process in which the native conformation of a protein is re-acquired. • Renaturation can occur quickly and spontaneously, and disulfide bonds are formed correctly. • If a protein is unfolded, it can refold to its correct structure placing the S-S bonds in the right orientation (adjacent to each other prior to formation), then the correct S-S bonds are reformed. • This is particularly true for small proteins. • What about large proteins? • Proproteins, which are the precursors of protein, large, and unfunctional, must be proteolytic cleaved to be functional, this cleavage destroys and changes its primary structure, so if these proteins get denaturized, they won’t refold (because the primary structure has been changed). • In conclusion, any protein with changed primary structure won’t be renatured after denaturation. The protein will refold Problem solvers: Chaperones • Folding of the protein is very important, it’s thermodynamically favored, and every protein should have its own tertiary structure after folding it. • Sometimes, proteins misfold, even some proteins require help to be folded. • Chaperones do the job; they assist/help other proteins to fold correctly or correct the misfolding. • These proteins bind to polypeptide chains and help them fold with the most energetically favorable folding pathway. • Chaperones also prevent the hydrophobic regions in newly synthesized protein chains from associating with each other to form protein aggregates (fibers, and this is very toxic to the cell) . Problem solvers: Chaperones • As seen in the figure, the polypeptide will get in the chaperone which will help it to fold correctly using ATP (so it has ATPase activity), after the hydrolyzing of ATP, the correctly folded polypeptide will go outside the chaperone. • Chaperones are also called heat shock proteins (HSPs), they consist of two subunits: • HSP70 • HSP40 • They are named heat shock because they can tolerate high temperatures without being unfolded. Many diseases are the result of defects in protein folding. The problem of misfolding • When proteins do not fold correctly, their internal hydrophobic regions become exposed and interact with other hydrophobic regions on other molecules, and form aggregates. Beta sheets instead of alpha helices. Outcome of protein misfolding • Partly folded or misfolded polypeptides or fragments may sometimes associate with similar chains to form aggregates. • Aggregates vary in size from soluble dimers and trimers up to insoluble fibrillar structures (amyloid). • Both soluble and insoluble aggregates can be toxic to cells. Prion disease • Striking examples of protein folding-related diseases are prion diseases, such as Creutzfeldt-Jacob disease (in humans), and mad cow disease (in cows), and scrapie (in sheep), eating one piece of a cow’s meat with this disease can infect the human. • Pathological conditions can result if a brain protein known to as prion protein (PrP) is misfolded into an incorrect form called PrPsc. • PrPC has a lot of α-helical conformation, but PrPsc has more β strands forming aggregates. Properly folded misfolded Very toxic to the brain, it will make the brain sponge-like, full of walls. The prion protein • The disease is caused by a transmissible agent • Abnormal protein can be acquired by • Infection • Inheritance • Spontaneously Alzheimer’s Disease • Not transmissible between individuals • Extracellular plaques of protein aggregates of a protein called tau and another known as amyloid peptides (Aβ) damage neurons. Formation of plaques When there’s mutation in alpha secretase, another abnormal cleavage pathway takes place. Amyloid Precursor Protein Won’t be cleaved Quaternary structure What is it? It’s the 3d structure of more one subunit • Aggregates are possible from dimer to dodecamers(12) or higher. • Each part of a protein with quaternary structure is called a subunit of that protein • Proteins are composed of more than one subunit. • They are oligomeric proteins (oligo = a few or small or short; mer = part or unit) • The spatial arrangement of subunits and the nature of their interactions. • Proteins made of • One subunit = monomer • Two subunits: dimer • The simplest: a homodimer • Three subunits: trimer • Four subunit: tetramer • hemoglobin • …etc • Each polypeptide chain is called a subunit. • Oligomeric proteins are made of multiple polypeptides that are • identical → homooligomers (homo = same), or • different → heterooligomers (hetero = different) • Oligomer sometimes refers to a multisubunit protein composed of identical subunits, whereas a multimer describes a protein made of many subunits of more than one type. • The quaternary structure of a protein consists of the following characteristics of its subunits: 1. Their number (dimer, trimer, tetramer… etc) 2. Their kind of the subunits; identical or non-identical in size or primary structures (amino acids sequence). 3. Their arrangement in three-dimensional space that stabilized the quaternary structure. 4. the interactions between the subunits that stabilize the quaternary structure (only non-covalent interactions). How are the subunits connected? • Sometimes subunits are disulfide-bonded together ( membrane proteins). :‫ كالم الدكتور‬،‫هذا بعارض كالم الدكتور‬ ً ‫وبناء عىل هذا‬ one subunit ‫بعتبهم‬ ‫ ر‬disulfide bond ‫ الرابطة بيناتهم‬polypeptide chains ‫إذا كان عندنا‬ .quaternary structure ‫بعتب ال رـبوتي‬ ‫ما ر‬ .‫ استثناء‬membrane proteins ‫نعتب الـ‬ ‫لكن ممكن ر‬ • Most globular proteins, noncovalent bonds stabilize interactions between subunits (hemoglobin). • Many proteins that contain more than one chain are NOT said to have a quaternary st Example of the quaternary structure is the hemoglobin molecule, it contain 4 subunits (tetramer), 2 beta and 2 alpha. Complex protein structures Holo- and apo-proteins • Proteins can be linked to non-protein groups and are known as conjugated proteins. • When a protein is conjugated to a non-protein group covalently, the nonprotein group is known as a prosthetic group and the protein known as a holoprotein. • If the non-protein component is removed, the protein is known as an apoprotein. Others • Lipoproeins: Proteins associated with lipids, such as LDL, HDL, Chylomicrons, VLDL… etc. • They are important in transporting lipids. • Phosphoproteins: proteins that are phosphorylated by kinases enzymes. • Thr, Try, and Ser will acquire the phosphate group, because their R groups contain hydroxyl group. • This phosphate group may activate or inhibit the protein. Others • Hemoproteins: proteins with heme • Heme: 4 pyrrole rings and iron atom attached to them. • Such as hemoglobin and cytochromes. • Nucleoproteins: proteins with a nucleic acid • Such as Spliceosomes. • Glycoproteins: proteins with carbohydrate groups Classes of glycoproteins • N-linked sugars • The amide nitrogen of the R-group of asparagine and glutamine. • O-linked sugars • The hydroxyl groups of either serine or threonine or tyrosine. • Occasionally to hydroxylysine