The Effect of Protein Structure and Function on Protein Evolution - Essay Example

? Contents The Study of Homology (Homology of Homologous studies) p.3 2. - ification systems for homologous proteins p.4 3. - History of the Protein p. 6 4. - Stanley Miller Experiment p. 7 5. - Evolutionary Theories. p. 9 6. - Folding (Chaperones) p. 11 7. - Tools of the Trade; X-ray Crystallography and NMR. p.13 - Summary p. 16 References. p. 17 Abstract: A meta-analysis covering sixty years of biochemical research into the history and evolution of both the study and reality of proteins was conducted. Distinctions where made for the description and categorization of changes within protein lineages to identify Orthologous and Paralogous change, with immunoglobulin as an example.Literature review is conducted concerning the most pertinent theories of rates of protein mutation down through geologic history. Areas where mutations are more likely and less likely are both discussed. Theories concerning the activity and origin of early proteins are discussed in addition to the relationship between ancient peptide chains and self-catalytic RNA that may have given rise to Earth's proteomes. Theories of the formation of the first ribosomes from coallescing RNA fragments is described, and the co-evolution between the early information systems of the pre-cellular world, and the beginnings of functional proteins. Stanley Miller and his experiment are explained in addition to earlier attempts to account for the origin of life. Oparin's theories of complex coacervation are compared to the Miller-Urey amino-acid synthesis apparatus.The physical construction of the experimental chamber for the Miller experiment is illustrated, and the implications are described in detail. The theory of duplication and subsequent mutation is explained as the prevailing hypothesis for the generation of large functional proteins and families of proteins, as well as providing a means for the conservation of a pre-existing beneficial function. The process of protein folding is described, as well as the distinction between two- and multi-step proteins for the purpose of assembly into final conformation. Lastly, the most important tools are described for the direct physical analysis of protein structure - X-ray Crystallography and Nuclear Magnetic Resonance Imaging. Contributions to the structural understanding of biomolecules are mentioned, including the discovery of Rosalind Franklin, and her work on determining the crystalline structure of DNA. A solid experimental and theoretical basis confirms the structure, function,evolution, and construction of proteins within the cell. Additional work is necessary for such issues as amino-acid chirality, as it pertains to the origin of life,and more experimental support is needed for the early evolution of proteins. A collection of the most relevant protein structural elements is included. 1 - Homology of homology. Similarities exist in the studies of putative patterns in evolutionary theory, whether the Investigator studies subjects at the organismal level, or the molecular. Principles of convergent,and divergent evolution exist in both scales. Patterns inherent to protein evolution can be shown to follow discrete principles not dissimilar to some of those found in the zoological studies at the organismal level. When studying the universe of possible protein configurations, it seems plausible that the total number of sequences allowable is theoretically infinite.1 Originally, it is theorized that the first truly functional proteins arose as supportive cofactors for the replication and catalytic function of RNA pre-cellular systems, as RNA is the only know information-transmitting biomolecule that possesses its own catalytic abilities. 2 The breadth of protein potential creates a pressing need for an understanding of the patterns of sequence and structural evolution. Intense study has gone towards the illumination of relationships peptide combinations and diversity may reveal across the web of life on Earth. Several methodologies have arisen in recent decades to both categorize and detect homologous relationships. Logic modalities are needed both to seek out possible examples of homology among amino-acid structures, as well as other means to disprove relationships which are statistically unlikely. 2 - Classifications Towards the purpose of defining homologous relationships, the term congruence can be used in reference to a shared pattern in size and shape.4 It is an obvious criterion, in reference primarily to the most apparent three-dimensional (quaternary) structure of the protein in question. Conjunction - or lack therof is a means to detect or disprove a homologous relationship. True homology requires a decision-tree of mutually-exclusive choices following common descent leading to anatomical and/or structural variances. If it is presumed that two different structural or functional features diverged from a common ancestral origin; both forms cannot simultaneously exist in the same native structure. 4 For example, if eyes arose from a light-sensitive spot on a protist, and evolved into all forms of eyes used by metazoans, then there should not exist an organism with one pair of eyes like those of mammals and another pair of arthropod compound eyes. Proteins exhibiting detectable identity in three-dimensional space would display congruence, and must be presumed related at least. Whereas, if it were theorized that a particular quaternary structure arose solely from a disulfide linkage at a specific sequence, an identical shape should not exist in the same protein without that same disulfide linkage. But the definitive test of course, must at some point shift to amino acid sequence analysis. In terms of similarities at the level of the sequences themselves, two classifications have been described: Orthologous sequences, and paralogous sequences. Orthologous refers to differences in sequence due to their origination in different species. Paralogous sequences can exist regardless of species, but express differences due to genetic duplications. Most proteins on Earth possess multiple domains that presumably arose through a duplication process. Approximately 80% of eukaryote proteins possess multiple domans 5 . Paralogous deviations can be conserved despite speciation events. Thus, paralogous sequence deviations may occur at highly variable rates, if and when they do, until selective pressure imposes constraints. 6 An example of a wide-spread protein with factors that illustrate both of these homological patterns of deviation would be immunoglobulin. The importance of Immunoglobulin in animal survival throughout geologic history is difficult to overstate. In its function as both cellular receptor and mediator of active immunity against microbial pathogens, the demand for its activity has remained undeniable in the ongoing biological arms-race of immunity versus disease; in this role it is found in the most primitive of animals.5 Immunoglobulin is the active agent that forms antibody-structures which target and neutralize the proliferation of viral invaders when they attempt to pass between cells in the host. In addition to regulatory roles as a cell-surface receptor, it is a key initiator of the Complement cascade reaction targeting the cell walls of infectious bacteria. 8, 9 Being of such universal importance, immunoglobulin is wide-spread and has become adapted for the needs of different species. Those familiar with immunology will recognize the importance of the CD protein subunits as the external apparatus employed by immune system T-cells in their role as mediators of immune-system processes. The C-set proteins exist in vertebrates only, whereas invertebrate animal species have two alternative domains as part of their own immunoglobulin molecules, I-set and V-set domains. 10 The CD domain found in vertebrates is paralogous; it arose through a genetic duplication process not limited to any one particular species, and is in fact shared by many species just as the I and V-set domains are themselves shared by many invertebrates. Within these categories, different species may possess specialized modifications of these subunits, which are likely to be orthologous changes, having arisen due to singular speciation events. History of the Protein - The most likely candidate for the process of modern protein diversity appears to be a pattern of accidental duplication errors followed by subsequent sequence mutations. This mechanism should allow for the maintenance of the original function of the first protein in question, while providing the available framework necessary for the addition of novel features. Natural selection thereby gains a opening to work upon the duplicate protein, and additional traits can be added over time as changes in activity brought on by the modified duplicate are combined with the original activity that has been retained.2 Subsequent duplications filtered this way through natural selection can build extensive coding regions that are transcribed into the large, three-dimensional structural elements critical to modern biochemical processes. When such duplications prove favorable the recurrence of the mutational event that brought them about is encouraged. 3 As stated above, pre-cellular RNA strands are a likely candidate as a template that brought the first true proteins into existence. Weak or non-existent error correction during RNA replication would often result in misreads as RNA catalyzed its own replication with assistance from early proteins. Low fidelity could eventually lead to higher-order protein structures available to natural selection by allowing independence of final form from what is encoded by the original RNA template. 2 Today, it is known among entities that rely solely on RNA, namely RNA viruses that they possess features which lend themselves to lower genomic stability: loose packing, little or no supercoiling, in addition to limited error-correction mechanisms. 6 But the exact schedule of events that might have brought about the origin of the ribosome, the crucial engine of protein translation, is among the deepest mysteries of biology. It appears that the most indispensable proteins were the translation factors. tRNA's can be bound to ribosomes without initiation or elongation factors depending on the ionization conditions of the media.11, 12 Under the proper environmental conditions, polypeptide synthesis can indeed occur without the higher level controls of a modern cell. The ribosome itself may have originated from small, catalytic subunits composed of pure RNA merged into larger confections as they melded with additional RNA structural elements. 14 It is known that RNA can form stem-loop structures even when cut away from larger RNA structures. Sub-elements of RNA are even capable of codon recognition. 11, 16, 17 - Stanley Miller Experiment At the University of Chicago in 1952, an experiment was conducted that would set the tone for decades of origin of life research. Biochemist Stanley Miller created a reaction chamber to test the possibility of the formation of organic compounds from inorganic precursors. He was influenced by the theories of J.B.S. Haldane, and Alexander Oparin, who believed in a primitive Earth with chemical conditions favorable to spontaneous synthesis of organic molecules. 21 Oparin in particular, pioneered a theory of coacervation of lipid molecules capable of rudimentary metabolism prior to the evolution of any sort of information-carrying molecule. The theory was deemed insufficient, with no known mechanism by which the coacervates could reproduce. 22 Water, methane, ammonia, and hydrogen were sealed inside a sterilized assembly of glass tubes. The tubes and a series of flasks where looped into each other, one flask half-filled with a liquid intended to replicate the chemical composition of Earth's early oceans. Another flask contained a pair of electrodes. Upon heat evaporation of the water in the first flask from a boiler element, current passed between the electrodes in order to simulate the effects of atmospheric lightning. The water was then cooled, allowing it to pass into a condensation column and collecting trap to carry it back into the first flask for an ongoing cycle. fig. 1 Stanley Miller's experimental apparatus. Methane, Ammonia, and Hydrogen are included in the 'primitive ocean' flask, the sparks from the power supply combined with subsequent condensation caused the synthesis of organic compounds, the most common of which was glycine. Organic compounds bound up 10-15% of the available carbon in the system after a week of continuous operation. Specifically, all 20 of the common amino acids were present in varying quantities, forming the building blocks of all proteins utilized by living cells. The significance of the experiment is difficult to overstate, but under these conditions, the amino acids formed were a racemic mixture; with both right and left-handed optical isomers. The Miller/Urey experiment does not address the subsequent organization of amino acids into homochiral isomers. But under these conditions, a steady flow of organic molecules would accumulate in a primitive ocean. Oparin contributed theories for the partial function of a pre-cellular structure, similar to the simple protoplasm envisioned by 19th century thinkers, Miller's experiment illuminated the means to achieve a steady supply of the building blocks of life. - Evolutionary Theories Inevitable constraints to random change do exist. Limitations in protein evolution as defined by conserved regions in structure despite orthologous deviations may be ameliorated by site-specific co-evolution. 4 Seemingly minor changes in one location may modify the 'landscape' of protein functionality to permit further changes in regions normally conserved; if the functional flaws of mutation in the conserved region were minimized by prior alterations. 24 By way of example; a breach in a hose should result in leakage; unless prior changes rerouted liquid flow away from that section of the hose before the breach. For similar reasons, anyone attempting to repair heavy machinery would be advised to reroute, or disable the machine's function prior to making any internal adjustments. A comprehensive view of these sequence modifications requires more than simply a discussion of the mechanics of the protein itself. Selection factors outside the protein's structure may determine whether a given configuration is reproductively favorable for the organism in question. The same protein could exhibit different survival outcomes based on the genomic location of the genes that encode for it. In addition, entirely different proteins may exhibit functionalities dependent on the activity of a separate protein. Further, replication fidelity can have long-term consequences on the viability of a particular sequence. 20 It is possible to trace protein lineages into both adjacent chains of co-evolving residues, 16 and spatially separated residues that match one another's evolutionary progress despite physical distance on the quaternary structure. 13 Upon a duplication event, different protein domains can gradually be adapted for an expansion of the original, monomeric function. There is also evidence that more subtle modifications are possible within promiscuous regions of the domain. Adjustments and selection events can fine-tune in some cases, a proteins' reaction to its corresponding ligand. Changes in promiscuous regions can alter whether and to what extent a ligand may bind to the protein in question, what effect that has on the subsequent protein, or even the identity of the substrate molecule that initiates conformational changes. 24 Promiscuous binding regions can also allow a protein to exist in an indeterminate state between various conformations, this has been found with certain antibodies. 25 The greater the flexibility in binding sites, the weaker the subsequent attachment to the ligand will become, and the converse - the stronger a protein's affinity, the less flexibility exists in terms of what substrate will trigger the bond. The more diversity in sequence for a group of proteins, the more functional diversity exists. 26 This adaptability seems just as crucial as the initial duplication mutational event towards propelling protein structures along an evolutionary destiny. In essence, the two vectors for measuring the rate of evolution are a function of time and distance. The number of actual sequence differences between a given group of orthologous proteins is divided by the total geologic time that separates a given speciation event on the organismal level. 19 Rates of change can vary not only between nucleotides, but also between different chromosomes. 22 Once proteins are identified as orthologous, the ancestral substitution site must be identified between the sequences as the starting point for measurement of changes. Once changes are underway, amino-acid substitution rates are balanced by the stability of the mutations once having occurred.19 There is evidence that up to 40% of the deviations observed in protein evolution are the result of varying mutation rates. 27 Resulting in cellular, organismal, and ecological factors that influence protein evolution entirely beyond the effects of the specific structural components of that protein. In terms of factors on the level of the organism, putative trends can be extrapolated that give additional predictive power for theories of protein evolution. Notable differences are apparent in terms of evolution based upon the proteins' connection to overall organ-system function. In humans, there is evidence that proteins connected to certain transcriptional regulatory sites and accompanying receptors have high rates of mutation and subsequent protein evolution. Highly conserved proteins are found controlling factors such as the actual metabolism of energy substrates such as glucose. Extreme divergence has been found between transcription factors controlling gluconeogenesis between invertebrates and primates. 15 A similar trend occurs in human insulin signalling, revealing genes with low conservation in terms of when and how a metabolic function occurs; but high conservation for the actual catalytic process itself. 30 Folding - On a more immediate level, the actual mechanics of the physical process by which proteins perform their function and are assembled are crucial issues to proteomics and molecular biology in general. During the assembly process, the rate constant and the kinetic order are the factors studied to give insight into the rate of protein folding. The rate constant itself is dependent on hierarchical factors, in large part the topology of the secondary structure, as it pertains to the formation of folds to comprise tertiary and quaternary structures, ? helixes appear to have minimal influence on folding kinetics. 31. Kinetic Order is the arrangement of intermediate stages required for the protein to reach its ultimate, three-dimensional shape. The order is classified as either two-state, or multistate kinetics. While the number of steps necessary for the folding process to complete is a factor, it is not necessarily the definite one. Variances in fold rates can range between milliseconds from up to an hour. 20 Present research suggests that the rate is largely dependent on native structure topology, more so than the actual amino-acid sequences. 18 Assisting with the folding process are a classification of gene products known as Chaperonins. Chaperon proteins is the name given to describe the heat-shock 60 family of protein, whose purpose is to assist with the potential hazards of protein folding. At higher temperatures and pressures it is probable that many proteins will misfold, and possibly form unbreakable aggregate bonds that prevent any useful function for the life of the molecules. Chaperonins largely act to prevent unproductive aggregation. A Co-chaperone, along with a does of ATP are necessary to prevent folding errors leading to clumping of peptides within the cell. Proteins with multi-state folding kinetics will collapse into a form that binds to the chaperon and reduces the tendency for aggregation, encouraging conformation with the correct structure. 31 In evolutionary terms, there is evidence that the selection of amino acids sequences in peptides and proteins exists to minimize the tendency to unfold and form disruptive aggregates inside the cell. Chaperones and precise choices in constituents are needed because folding and aggregating are closely related processes. In addition to pH and temperature, the outer surface of the protein structure can effect the physicochemical 'landscape' upon whih the conformation of the protein is determined. These outer surfaces too, can influence protein folding over aggregation. The dangers of aggregation specifically is one of toxicity. Whether inside or outside a cell, aggregates can trigger apoptosis, or simple necrosis.14 When improperly folded, or unfolded - agrregate-prone regions of the inner core of a complex protein can be exposed under the right condition of pH, which can modify the dielectric constant, causing a softening of structure enough for the inner regions to be exposed 17 For membranes, lipid density, compactness, curvature, fluidity and rigidity can also be factors in the electrostatic potential and hydrophobicity of a protein. Certain fold-types can yield clues in terms of the ultimate function of the protein. The circular, barrel-like configuration ringed by helices, known as triosephosphate isomerase, or the TIM barrel often catalyzes enymatic reactions; whereas several protein types display fold patterns similar to the characteristic structure of immunoglobulin, where they serve as signaling transducers. 32 ,33 , 34, 35 Tools of the Trade - X-ray Crystallography X-ray crystallography is among the best tools in the biologist's arsenal to determine the exact structure of a protein, or indeed inorganic forms as well - with a wide range of elements able to crystallize, including most transition metals, minerals, and semi-conductors It is generally considered the method of choice for examining novel atomic structures. The arrangement of atoms in a crystal can be inferred from a beam of X-rays striking a crystal and subsequently diffracting into different directions. 38, 39 The size of atoms, as well as the characteristics of chemical bonds can be determined by the patterns of X-ray scattering. Though in ligand-activated proteins of high conformational complexity, additional time may be needed to derive the full three-dimensional structure. 39 The technique provides information that can reveal details concerning a variety of unusual physical properties of a substance. The bonding patterns of octahedral metal atoms (aluminum hexachloroplatinate) and diamonds were revealed with this method. 40 In 1923, the structure of the first organic compound, hexamethylenetetramine was revealed via this method. 39 Far larger structures of true proteins were resolved later in the 1950's when the structure of sperm whale myoglobin was solved. 42 Today, X-ray crystallography can be used to resolve questions of how novel drugs will interact with specific proteins and the most likely strategies to improve those interactions. Certain molecular receptors are a challenge for crystallography, because many embedded membrane proteins require emulsifiers or detergents to dissolve them into liquid, and these agents may impede the process of crystal formation. Prior to this test, most proteins are grown in solution though an incremental reduction in molecular solubility. The conditions needed for protein growth are both nucleation, and subsequent growth. The factors that allow the condensation of a structure around a solid nuclei may differ from the conditions that will allow the most subsequent growth. The crystallographer may need multiple solutions, in order to favor the formation of a single, large crystal, and then in the growth phase, to ensure that only a single crystal forms per droplet.43 When the crystals have been grown, the diffraction occurs. The most precise method is single crystal X-ray diffraction, where the beam strikes a single crystal, resulting in scattered beams. A photo- detector, such as film, will surround the crystal, resulting in a speckled pattern of diffracted spots. As the crystal rotates, the intensity and direction of the beams can be recorded as a reflection. These reflections originate from regularly spaced planes within the crystal structure; the exact shape and position of the atoms can then be inferred. 44 - Other techniques include powder diffraction, and X-ray fiber diffraction, used by Rosalind Franklin in the determination of the double-helical structure of the DNA molecule.45 These techniques typically offer less information than single-crystal diffraction, but the large crystal required is more difficult to acquire. Figure 2: The spot pattern resulting from diffraction corresponds to hard planes within the molecular structure of the crystal in question. This pattern is then translated into an electron density map and eventually an atomic model. Protein nuclear magnetic resonance spectroscopy - It has been known since 1944 that magnetic nuclei have the ability to absorb radio frequenct energy if placed in a magnetic field of a strength matching the identity of the nuclei. When this absorption occurs, the nuclei is said to be in resonance. Difference resonance frequencies are produced by different atomic nuclei, allowing the trained investigator to determine useful, structural and chemical data pertinent to the molecule in question. 46 The theory of magnetic resonance holds that any nuclear particle will have a spin. Even numbers of protons and neutrons in an atom will exhibit a spin that cancels itself out, resulting in zero overall spin. The technology finds and measures spin differences where an imbalance exists. 47, 48 By measuring peaks in the resonance spectra, it is possible to derive the identities of a variety of molecules. It is even possible to make distinctions between molecules that differ only in their local environment. 48 While less popular in the field of protein analysis that X-ray crystallography, NMR can provide detailed information about the internal composition of matter in a variety of phases, and is projected to lead to advancements in quantum computing in the future. 49, 50 Summary - The study of homology, as it pertains to proteomics has been defined, in addition to the terminology of orthologous and paralogous shifts in sequence over time. This provide a basis for classifying homology throughtout speciation events as it pertains to proteins altered within their respective lineages, as well as proteins largely conserved throughout phyla. The mechanisms of protein descent, function, and construction have been described, in addition to the standard mechanism by which protein development and evolution progress via duplication-mutation. 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