RPI-BIOL-2120 Lecture 5: Protein Structure & Function PDF

BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 5 PROTEIN STRUCTURE & FUNCTION Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 4...

BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 5 PROTEIN STRUCTURE & FUNCTION Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 4 Images provided by M. T. Klein or W. W. Norton & Company unless stated otherwise MTK, 2025-01-06 (1)  OVERVIEW  Biological activity is mostly driven by the interactions between proteins  Other biological macromolecules obviously play important roles in life: Carbohydrates store energy and act as structural elements Lipids store energy, provide cushioning in the form of adipose tissue, and are the fundamental component of cell membranes DNA stores genetic information; RNA has a host of different functions including a central role in gene expression (mRNA)  But proteins offer a dynamism not seen in these other molecules  When thinking about the functions of proteins and how these functions are regulated, never forget the tight correlation between a macromolecule’s structure and its function; this is of particular importance when considering the myriad of functions proteins play in biology MTK, 2025-01-06 (2)  WHY DID RNA LOSE ITS JOB?  In the pre-life RNA world of early Earth, what characteristics of RNA made it possible for this type of macromolecule to fulfill the roles of proteins?  What properties of proteins make them better suited for these roles than RNA? MTK, 2025-01-06 (3)  AMINO ACIDS  The shape of a protein is fundamentally specified by its amino acid sequence  Covered in the previous lecture: Amino acids are the monomers that are used to construct polypeptides They are organic molecules with carboxyl and amino groups Amino acids differ in their properties due to differing side chains, called R groups 19 of the 20 standard amino acids have chiral centers at their α carbon (glycine being the exception) What is the term used to describe ꟷ Polypeptides are only built from L-isomers (left-handed isomers) a molecule with both acidic and basic functional groups? ꟷ D-isomers are rarely used in nature overall zwitterionic MTK, 2025-01-06 (4)  AMINO ACIDS NONPOLAR SIDE CHAINS  Nonpolar side chains make these amino acids more hydrophobic When incorporated into a polypeptide, these amino acid residues will orient their side chains away from water, usually towards the core of the protein or towards a surface of the protein that will interact with the hydrophobic tails of membrane phospholipids MTK, 2025-01-06 You should be able to recognize that these amino acids are hydrophobic (5)  AMINO ACIDS NONPOLAR SIDE CHAINS  Highlights: Glycine is the only non-chiral amino acid (it lacks a side chain); the bonds at the α-carbon can freely rotate allowing increased rotational flexibility in the polypeptide at this residue Proline is the only amino acid that forms a ring structure with the backbone, reducing flexibility in this region of the polypeptide Methinomine (1) is the starting amino acid in every polypeptide unless cleaved off after synthesis, (2) is found in eukaryotic and archaeal polypeptides but not bacterial ones (they use f-methionine instead) and (3) contains sulfur but cannot form disulfide bridges Be able to recognize the structures of glycine and proline MTK, 2025-01-06 (6)  AMINO ACIDS POLAR SIDE CHAINS  These amino acids have polar bonds somewhere in their side chains, making them hydrophilic; however, they will not be ionized in the environment of the cell This means they will form hydrogen bonds with water and be attracted or repelled by charged atoms and dipoles But they will not form ionic bonds with other side chains You should be able to recognize that these amino acids are hydrophilic Be able to recognize the structure of cysteine (the thiol group is important) MTK, 2025-01-06 (7)  AMINO ACIDS POLAR SIDE CHAINS  Highlights: Cysteine contains a thiol group (-SH) and can form disulfide bridges (covalent bonds) with the thiol groups of other cysteine residues – how do you think this will affect protein structure? Asparagine and glutamine are similar to aspartate and glutamate – what’s the difference? Serine, threonine, and tyrosine are commonly phosphorylated, which acts are a molecular on/off switch for the protein – why would the addition of a phosphate group to these residues affect the protein’s function? You should be able to recognize that these amino acids are hydrophilic Be able to recognize the structure of cysteine (the thiol group is important) MTK, 2025-01-06 (8)  AMINO ACIDS ACIDIC SIDE CHAINS  These amino acids have an additional carboxylic group terminating their side chains But this does not mean a polypeptide can become branched at this point during synthesis  Within the environment of the cell, these side chains will have a negative charge and be able to form ionic bonds with positively charged (basic) amino acid residues and other ionized molecules  They will also enhance the water solubility of the polypeptide as they are able to interact with the positive dipoles of water and other molecules  Both can be phosphorylated Be able to recognize these amino acids as having acidic side chains MTK, 2025-01-06 (9)  AMINO ACIDS BASIC SIDE CHAINS  These amino acids have an additional nitrogenous groups as part of their side chains Again, this does not mean a polypeptide can become branched at this point during synthesis  Like with acidic side chains, these will have the capability to form ionic bonds and dipole interactions  They will also enhance the water solubility of the polypeptide  All three can be phosphorylated Be able to recognize these amino acids as having basic side chains MTK, 2025-01-06 (10)  LEVELS OF PROTEIN STRUCTURE  As they’re being synthesized, polypeptides fold into their lowest energy conformation Think about trying to hold two attracting magnets apart – this takes energy You expend less energy if you release the magnets and let them collide This is similar to how different portions of a protein (domains) will attract https://ars.els-cdn.com/content/image/1-s2.0-S0014579301024863-gr1.jpg each other – the same is true for repulsion How does the presence of water affect conformation?  Misfolding of a protein can be devastating Function of the protein will likely be lost The protein could become toxic to cells or interfere with normal physiology – Alzheimer's Disease, Parkinson’s Disease, Huntington’s Disease, and infectious prion diseases are all cause by misfolded proteins MTK, 2025-01-06 (11)  LEVELS OF PROTEIN STRUCTURE PRIMARY STRUCTURE  Primary structure refers to the amino acid sequence of a polypeptide This sequence is encoded in DNA (genes), which are transcribed into mRNA, then translated into a specific polypeptide sequence at ribosomes  The joining of two amino acids forms a covalent bond called the peptide bind between the carbon of the carboxylic group of the first amino acid and the nitrogen of the amino group of the second one  The linking of two amino acids yields a dipeptide  The linking of 3 or more yields an oligopeptide (few residues) or polypeptide (many residues) MTK, 2025-01-06 (12)  LEVELS OF PROTEIN STRUCTURE PRIMARY STRUCTURE  Note the alternating position of the side chains  Why is this the favored arrangement of side chains? Think in 3 dimensions MTK, 2025-01-06 (13)  LEVELS OF PROTEIN STRUCTURE PRIMARY STRUCTURE  At ribosomes, additional amino acids are added to the carboxyl group of the growing polypeptide chain The polypeptide begins with the N-terminus (the amino-terminus) It ends with the C-terminus (the carboxyl-terminus, carboxyl-tail)  Some parts of a polypeptide my not have any structure above this and are called unstructured regions These regions tend to be flexible parts of the polypeptide and are found between structured regions, allowing the protein to fold properly MTK, 2025-01-06 (14)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone  The α helix and the β pleated sheet (or simply just β sheet) are the dominant folding patterns Why do you think that secondary structure usually does not vary much? MTK, 2025-01-06 (15)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  A closer look at the α helix The stacking of similar subunits in a regular way will yield a helical structure α helices are always right-handed MTK, 2025-01-06 (16)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  A closer look at the α helix Many membrane-bound proteins cross the lipid bilayer as an α helix This is the dominant patter for membrane bound enzymes and receptor proteins MTK, 2025-01-06 (17)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  A closer look at the α helix Intertwined α helices can form a stiff coiled-coil Can you think of any proteins that are constructed like this? MTK, 2025-01-06 (18)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  A closer look at the β sheet β sheets form rigid structures at the core of many proteins Antiparallel β sheets are most common but parallel β sheets are also present MTK, 2025-01-06 (19)  LEVELS OF PROTEIN STRUCTURE SECONDARY STRUCTURE  A closer look at the β sheet β sheets can stack to form an amyloid structure This structure resembles the type of insoluble aggregates observed in the neurons of individuals with different neurodegenerative diseases MTK, 2025-01-06 (20)  LEVELS OF PROTEIN STRUCTURE TERTIARY STRUCTURE  The tertiary level of structure is determined by interactions mainly between the side chains of the amino acid residues; interactions between side chains and the backbone are also common  These interactions include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions  Covalent bonds between cysteine residues called disulfide bridges reinforce the structure of some proteins like antibodies – what would happen to this type of protein if you boiled it? MTK, 2025-01-06 (21)  LEVELS OF PROTEIN STRUCTURE TERTIARY STRUCTURE  Hydrophobic forces help proteins fold into compact conformations  In a folded protein, polar amino acid side chains tend to be positioned on the surface and interact with water; nonpolar amino acid side chains are buried on the inside to form a tightly packed hydrophobic core of atoms that is hidden from water MTK, 2025-01-06 (22)  LEVELS OF PROTEIN STRUCTURE TERTIARY STRUCTURE  Many polypeptides are composed of different functional domains Not to be confused with proteins made of different polypeptide subunits (i.e., quaternary structure)  Elements of secondary structure pack together into stable, independently folded, globular elements called domains  The domains are often linked by unstructured segments of the polypeptide MTK, 2025-01-06 (23)  LEVELS OF PROTEIN STRUCTURE QUATERNARY STRUCTURE  Some proteins are composed of a single polypeptide, but those that are composed of two or more polypeptides are said to have quaternary structure  Examples: Collagen is a fibrous protein consisting of three polypeptides coiled like a rope Hemoglobin is a globular protein consisting of four polypeptides: two alpha subunits and two beta subunits Hemoglobin MTK, 2025-01-06 (24)  PROPER PROTEIN FOLDING  Polypeptides begin to fold into their most thermodynamically sable conformation as they’re being synthesized; however, this conformation may not be an active one  Helper proteins called chaperones are often needed to aid in proper folding of a polypeptide as it’s extruded from a ribosome; chaperones will prevent misfolding and ferry the polypeptide to chaperonin  Chaperonin is a complex structure that will accept a newly synthesized polypeptide into an inner chamber; chaperonin will change shape, contorting the polypeptide within, giving it a new stable conformation Video: https://drive.google.com/file/d/1om4s_Q0S2xxwrDwrO1fKJIyKqmr6dIVe/view?usp=sharing MTK, 2025-01-06 (25)  PROTEIN MISFOLDING  Misfolded proteins can result in devastating outcomes (see earlier slide about amyloid structures)  Prions are proteinaceous infectious agents – they are misfolded versions of normal proteins found in the brains of mammals Cellular-PrP is the healthy conformation; its functional domain is composed of α helices Prion-PrP is the disease-causing conformation; it has β sheets in an amyloid arrangement Prion-PrP causes cellular-PrP to refold into prion-PrP, resulting in an exponential growth of prion-PrP in the brain MTK, 2025-01-06 (26)  PROTEIN MISFOLDING  The amyloid structure of prion PrP is difficult to disrupt and will form aggregates in the brain, causing inflammation and degradation of brain tissue resulting in a condition called spongiform encephalopathy – this condition is always terminal  Prions are infectious and can be transmitted via blood transfusions (rare), corneal transplants, ingestion of contaminated meats, and possibly other modes – there is no cure and standard sterilization methods are ineffective MTK, 2025-01-06 (27)  DETERMINING PROTEIN STRUCTURE  Proteins come in a wide variety of shapes and sizes Each polypeptide to the right is shown at the same scale  Determining the structure of a protein is not a trivial matter Knowing the amino acid sequence of a polypeptide can help inform us about the type of protein it will form We can also make some simple predictions about structure (e.g., the presence of α helices) Beyond this, it becomes much more laborious to predict or directly assess the structure of a protein MTK, 2025-01-06 (28)  DETERMINING PROTEIN STRUCTURE  X-ray crystallography is often used to determine a protein’s structure  For hard-to-crystalize proteins, much of our knowledge of their structure has historically been inferred by their primary sequence What kinds of proteins might be hard to crystalize?  Another method is nuclear magnetic resonance (NMR) spectroscopy, which does not require protein crystallization but provides far less information  Bioinformatics uses a computational approach to predict protein structure from amino acid sequences; this process is complex and requires significant computing power (https://foldingathome.org/) Artificial intelligence has become a major force in the world of bioinformatics, reducing compute times by several orders of magnitude in many cases MTK, 2025-01-06 (29)  DETERMINING PROTEIN STRUCTURE PDF: https://drive.google.com/open?id=11xzCiDtXnzEpsYNN8yK_y47T-gS4d0_X MTK, 2025-01-06 (30)  EXTRA CREDIT DISCUSSION: PROTEIN STRUCTURE USE BLACKBOARD DISCUSSION BOARD TO POST ANSWERS  Find a peer-reviewed research article (https://pubmed.ncbi.nlm.nih.gov/) that investigates a protein’s structure. Give the title of the article, year of publication, journal, and authors. Provide a link to the article. What protein was investigated? Give some background as to what this protein does in nature and why understanding its structure is important. What techniques were used to predict or assess its structure? How is this protein’s structure related to its function? MTK, 2025-01-06 (31)  QUICK REFERENCE AMINO ACID GROUPED BY SIDE CHAIN MTK, 2025-01-06 (32)  QUICK REFERENCE LEVELS OF PROTEIN STRUCTURE MTK, 2025-01-06 (33)  QUICK REFERENCE EXAMPLES OF SOME GENERAL PROTEIN MTK, 2025-01-06 FUNCTIONS (34)