Chapter 3- Proteins: Composition and Structure Flashcards
Why are proteins so important?
Excluding water and fat, our bodies are made up almost entirely of proteins- proteins are the main component of muscles, bones, organs, and skin. Proteins perform different essential dynamic and structural functions in mammals including catalysis of chemical transformations, transport, metabolic control, and contraction. The number of proteins simultaneously expressed in a single human cell ranges between 20,000 and 50,000. Many drugs and toxic compounds are transported bound to proteins.
Enzymes
Proteins that catalyze chemical reactions. Almost all of the chemical reactions in living organisms require a specific enzyme catalyst. The catalyst ensures that the reactions occur at a rate that is compatible with human life- not too fast and not too slow
Proteins and genetic diseases
Many genetic diseases result from altered levels of enzyme production or alterations to their amino acid sequence. About 1/3 of human genes code for enzyme proteins.
Hormones and proteins
Hormones: Insulin, thyrotropin, somatotropin, prolactin, luteinizing hormone, and follicle-stimulating hormone are proteins.
Protein cell signaling function
Proteins bind and carry lipids, metabolites, steroid hormones, vitamins, signaling molecules, and minerals from their sites of synthesis to their sites of action/elimination.
Peptides vs proteins
Many polypeptide hormones have a low molecular weight (<5 kDa) and are referred to as peptides. In general, the term protein is used for molecules that contain over 50 amino acids and peptide is used for those < 50 amino acids.
Protein functions in transcription/translation (5)
- Histone proteins are associated with DNA
- Repressor and enhancer are transcription factors that control gene transcription
- Proteins regulate DNA structure
- Proteins transcribe DNA into RNA. During transcription, DNA serves as a template for complementary base pairing, catalyzed by RNA polymerase 2, which forms mRNA
- Transcription and translation of the DNA code results in the joining together of amino acids into a specific linear sequence characteristic of a protein.
General structure of amino acids
Amino acids consist of a central (alpha) carbon, which is bonded to an amino group, carboxyl group, hydrogen atom, and side chain (R). Each of the 20 amino acids contain different side chains- side chains can be polar, hydrophobic, and have other properties
Why is the primary structure of proteins important?
Primary structure of protein is required to understand its function and mechanism of action, its biosynthesis and its relationship to other proteins with similar physiological roles: e.g.: Insulin. Proteins are also compared by sequence identity rather than just structure-
proteins will likely have the same function if they have 70-80% sequence identity. If their similarity is more than 90%, the proteins are likely from the same family
Why is correct protein folding important?
Correct protein folding is required for correct function. Specific families of proteins have similar folding/structure and therefore carry out similar functions
Proinsulin
Proinsulin is produced in pancreatic islet cells as a single polypeptide chain containing 86 amino acids and 3 intrachain cysteine disulfide bonds. Insulin is initially synthesized as proinsulin, and it is transformed into biologically active insulin before secretion from the islet cells
How is proinsulin transformed into insulin?
Proinsulin is transformed into biologically active insulin by proteolytic cleavage prior to its separation from islet cells. Cleaved by proteases present in the islet cells between residues 30-31 and 65-66 releasing two molecules, a 35 residue fragment (C-peptide) and insulin, which consists of two polypeptide chains (A and B).
Homologous proteins
Sequence comparisons or alignment are commonly used to predict the similarity in structure and function between proteins. Two sequences are homologous when their sequences align. The comparison of the primary structures of insulins from different animal species shows the residues essential and nonessential to its hormonal function.
Similarity of insulin from different species
The aligned primary structures of insulin have identical residues in most amino acid positions except for residues 8, 9, and 10 of the A chain and residue 30 of the B chain. Other residues are rarely substituted, suggesting that they have an essential role in function, or that they are conserved. Insulin from different species is also very similar to human proteins. Their amino acid sequences only differ by around 20%, and they have the same function as human proteins. Prior to the use of human insulin, animal insulin was used for diabetics
Non-conserved residue substitution
A non-conserved residue substitution involves replacement of an amino acid by another amino acid of different polarity (Polar residue vs nonpolar residue).
What insulin was used prior to the development of human insulin?
Prior to the development of recombinant human insulin, both porcine and bovine insulins were used in human diabetics to treat diabetes- this is still used in countries that don’t have access to human insulin. Because of differences in sequence from human insulin, some diabetic individuals will have an initial allergic response as their immunological system recognizes the insulin as foreign. However, the frequency of a deleterious immunological response to pig and cow insulins is small; the great majority of the population is able to use these insulins without complication. Human insulin is the primary insulin used in developed countries. It is developed from genetically engineered recombinant bacteria (like E. coli)
Why can many people use pig and cow insulin without complications?
This is due to the small number of amino acid sequence changes between the species and the fact that they do not significantly change the insulin structure than that of human insulin. Pig insulin is more acceptable than cow in insulin-reactive individuals with more sequence similarity to human insulin.
Plasma lipoproteins
Plasma lipoproteins are complexes of proteins and lipids and the lipoprotein particles function to transport lipids from tissue to tissue and participate in lipid metabolism. Four classes exist in the plasma of normal fasting humans as distinguished by their density (high, low, intermediate, and very low density). Their protein components are termed as apolipoproteins, and each class of lipoprotein has a characteristic apolipoprotein composition.
Apolipoproteins
The protein components of plasma lipoproteins (lipid and protein complexes. Each class of lipoprotein has a characteristic apolipoprotein composition.
The most prominent apolipoproteins are
1. apolipoprotein ApoA in high-density lipoproteins (HDLs),
2. ApoB in LDLs, IDLs, and VLDLs 3. ApoC in IDLs and VLDLs.
Each apolipoprotein class is genetically and structurally different
Hyperlipoproteinemia
Disorders of the rates of synthesis or clearance of lipoproteins from the bloodstream. Detected by measuring plasma triacylglycerol and cholesterol. There are 5 types. Hypothyroidism can produce a very similar hyperlipoproteinemia. These patients have an increased risk of atherosclerosis.
Type 1 Hyperlipoproteinemia
ApoC deficiency – patients have very high plasma triacylglycerol concentrations and suffer from xanthomas and pancreatitis. Xanthomas are yellow colored deposits on the skin
Type 2 Hyperlipoproteinemia
Elevated LDL levels – due to defects in LDL receptors.
Homozygous patients often have very high LDL levels and may have myocardial infarctions
Type 3 Hyperlipoproteinemia
Due to abnormalities of ApoE protein- the protein can’t function properly
Type 4 Hyperlipoproteinemia
Very common, VLDL levels are increased due to obesity, diabetes or alcohol.
Type 5 Hyperlipoproteinemia
This hyperlipoproteinemia is, like type I, associated with high triacylglycerol levels, xanthomas and pancreatitis.
Abetalipoproteinemia
A genetic disease that is characterized by absence of VLDLs and LDLs due to an inability to synthesize apolipoproteins (ApoB). - accumulation of lipid droplets in the cells of the small intestine, malabsorption of fat, acanthocytosis (spiny-shaped red cells), and neurological disease (retinitis pigmentosa, ataxia, and retardation).
Tangier disease
An alpha-lipoprotein deficiency, and is a rare autosomal recessive disease in which the HDL level is 1–5% of its normal value. Clinical features are due to the accumulation of cholesterol, which may lead to hepatomegaly and splenomegaly. The plasma cholesterol and phospholipids are greatly reduced.
Spontaneous protein folding
The ability of a primary protein structure to fold spontaneously to its native conformation, without any information other than the amino acid sequence itself, has been demonstrated with many proteins. Such observations led to the hypothesis that the amino acid sequence contains the information for spontaneous folding to its unique active conformation under correct solvent conditions.
How is protein folding initiated?
There is evidence that folding is initiated by short-range noncovalent interactions between a side chain and its nearest neighbors (other side chains of different amino acids). Particular side chains have a propensity to promote the formation of α-helices, β-strands, and loops. The amino acid sequence of a protein may contain information for its spontaneous folding. Additionally, cofactors or prosthetic groups may help the protein to spontaneously fold, and chaperone protein can assist with this process and make sure the protein is folding normally
Bonding forces in a protein
Noncovalent forces (weak bonding forces) cause a polypeptide to fold into a unique native conformation and then stabilize the native structure against denaturation. Van der Waals forces are the weakest bonding forces, covalent bonding forces are the strongest
Prion diseases
A prion is a type of protein that can trigger normal proteins in the brain to fold abnormally. Several fatal and transmissible neurodegenerative diseases in humans and other animals, spread by consumption of infected meat products. Characterized by ataxia, dementia, and paralysis and is almost always fatal. Pathological examination of the brain shows amyloid plaques and neurodegeneration. Misfolded proteins will have abnormal electrical activity in the cell
Transmission of prion diseases
Can be spread by consumption of infected meat products. If the animal that the meat came from was infected, the infection may be transmitted to humans. This occurs in mad cow disease when humans acquire the prion form the ingestion of bovine prions in contaminated meat from cattle. This infectious disease can also appear spontaneously or due to the inheritance of a mutated prion protein gene.
Why do prions cause issues in the cell?
The highly soluble cellular conformation of the prion protein is converted to the insoluble toxic conformation. In several neurodegenerative diseases similar conformational equilibriums occur between a soluble predominantly α-helical protein conformation and a less soluble β-strands conformation, with the β-strand conformation polymerizing into insoluble amyloid fibrils. The plaques also have abnormal electrical activity in the cell.
Prion plaque structure
The protein unit differs with the disease type, but the plaque formed all have a similar amyloid fibril structure. - Alzheimer, Parkinson, Huntington, and amyotrophic lateral sclerosis (ALS, Lou Gehrig). The structure varies since different proteins are associated with different diseases
3 different routes of initiation of amyloid formation
- Conformational transformation of the normal prion protein to the insoluble form.
- Infective proteins introduced through ingestion of contaminated meat.
- Inheritance of a mutated prion gene with ability to fold into insoluble form.
Inherited phenotypes of prion disease
Inherited phenotypes include Gerstmann–Sträussler–Scheinker syndrome and fatal familial insomnia. Sporadic (or spontaneous) disease results in Creutzfeldt–Jakob Disease (CJD) and is typically fatal within 1 year of the onset of illness.
Symptoms- ataxia, poor balance, gait changes, poor coordination, difficulty walking. Mad cow disease, as one example, is fatal within one year of onset of symptoms
Amino acid analysis in the diagnosis of disease
Elevated concentrations of amino acids are found in plasma or urine in a number of clinical disorders. An abnormally high concentration in urine is called an aminoaciduria.
2 methods of protein separation based on charge
- Electrophoresis- isoelectric focusing and capillary electrophoresis
- Ion-exchange column chromatography
Electrophoresis
The protein is dissolved in a buffer solution at a particular pH is placed in an electric field (Polyacrylamide, Agarose). Depending on the relationship of the buffer pH to the pI (Isoelectric Point, net charge of protein is zero) of the protein, the protein moves toward the cathode (-) or the anode (+) or remains stationary (when pH = pI).
Ion-exchange column chromatography
Includes resin consisting of insoluble materials (agarose, cellulose) that contain charged groups. Negatively charged resins bind positively charged molecules/proteins and are cation-exchange resins. Positively charged resins bind anions strongly and are anion-exchange resins. Molecules with the same charge as the resin are eluted first, followed by those with an opposite charge to that of the resin. An increasing gradient of ionic strength also decreases the charge interactions, as the ions compete with the proteins for binding.
