Protein Structure: Unveiling Molecular Dynamics and Interactions in Biological Macromolecules

Chapter 1: Protein structure

Structure of a protein refers to the three-dimensional arrangement of atoms within a molecule that is composed of amino acid chains. The sequences of amino acids, which are the monomers of the polymer, are the building blocks from which proteins are constructed. Proteins are polymers, more precisely polypeptides.  A single monomer of amino acids can also be referred to as a residue, which is a term that describes a unit of a polymer that repeats itself. In the process of forming proteins, amino acids go through condensation reactions. During these events, the amino acids lose one water molecule at a time in order to acquire a peptide bond and therefore link themselves to one another. As a matter of tradition, a chain that contains fewer than thirty amino acids is typically classified as a peptide rather than a protein. Proteins fold into one or more precise spatial conformations in order to carry out their biological activity. These conformations are driven by a range of non-covalent interactions, including hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing, among others. It is frequently required to identify the three-dimensional structure of proteins in order to have an understanding of the tasks that proteins perform on a molecular level. This is the subject matter of the scientific discipline known as structural biology, which is concerned with determining the structure of proteins by the utilization of methods like as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, cryo-electron microscopy (cryo-EM), and dual polarization interferometry.

The number of amino acids that make up a protein structure can range anywhere from tens to several thousand. With a physical size ranging from one to one hundred nanometers, proteins are categorized as nanoparticles. Protein subunits have the potential to be assembled into extremely massive protein complexes. One example is the formation of a microfilament, which is comprised of several thousands of actin molecules.

During the process of carrying out its biological activity, a protein will typically go through structural modifications that are reversible. The transitions that occur between the many conformations of a protein are referred to as conformational alterations. Different conformations are the alternative configurations that can be found within the same protein.

The structure of proteins can be broken down into four separate layers.

The progression of amino acids along the polypeptide chain is what is meant by the term primary structure when referring to a protein. A process known as protein biosynthesis is responsible for the formation of peptide bonds, which are responsible for holding the main structure together. The carboxyl terminus (C-terminus) and the amino terminus (N-terminus) are the names given to the two endpoints of the polypeptide chain. These names are derived from the distinct characteristics of the free group that is present at either extremity. Counting of residues always begins at the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. This is the end where the peptide bond formation occurs. Proteins are characterized by their primary structures, which are defined by the genes that correspond to those proteins. Over the course of a process known as translation, a particular sequence of nucleotides found in DNA is converted into messenger RNA (mRNA), which is then read by the ribosome. Frederick Sanger was the one who laid the groundwork for the discovery of the sequence of amino acids in insulin, which established that proteins have characteristic sequences of amino acids. A protein's sequence is unmatched by any other protein, and it is responsible for determining both the structure and the function of the protein. Methods such as Edman degradation and tandem mass spectrometry are examples of techniques that can be utilized to ascertain the sequence of a protein. On the other hand, it is frequently read straight from the sequence of the gene by making use of the genetic code. When addressing proteins, it is strongly suggested that the term amino acid residues be utilized. This is owing to the fact that the formation of a peptide bond results in the loss of a water molecule, and as a result, proteins are composed of amino acid residues. It is not possible to read post-translational modifications from the gene because they are typically believed to be a part of the primary structure. Examples of such modifications include phosphorylations and glycosylations. As an illustration, insulin is made up of 51 amino acids that are arranged in two chains. While one chain contains 31 amino acids, the other chain only contains 20 amino acids.

When we talk about secondary structure, we are referring to highly regular local sub-structures that are located on the real polypeptide backbone chain. The α-helix and the β-strand or β-sheets are the two primary forms of secondary structures that were proposed by Linus Pauling in the year 1951. The patterns of hydrogen bonds that exist between the main-chain peptide groups are what define these secondary structures. The geometry of these objects is regular, as they are limited to particular values of the dihedral angles ψ and φ on the Ramachandran plot. When it comes to saturating all of the hydrogen bond donors and acceptors in the peptide backbone, the α-helix and the β-sheet are equivalent representations of this process. Although there are some components of the protein that are organized, there are no regular shapes that they form. Different from random coil, which is an unfolded polypeptide chain that does not have any definite three-dimensional structure, they should not be confused with random coil. It is possible for a supersecondary unit to be formed by a number of successive secondary structures.

Tertiary structure is the three-dimensional structure that is produced by a single protein molecule, which is more commonly referred to as a single polypeptide chain. It is possible for it to comprise one or more domains. In order to create a tight globular structure, the α-helices and β-pleated-sheets are effectively folded. Despite the fact that the folding is driven by non-specific hydrophobic interactions and the burial of hydrophobic residues from water, the structure is only stable when the components of a protein domain are locked into place by specific tertiary interactions. These interactions include salt bridges, hydrogen bonds, and the tight packing of side chains and disulfide bonds. Because the cytosol, which is the fluid that is found inside the cell, is often a reducing environment, disulfide bonds are something that are relatively uncommon in cytosolic proteins.

The quaternary structure is a three-dimensional structure that is made up of the aggregation of two or more separate polypeptide chains, also known as subunits, that function as a single functional unit, also known as a multimer. The non-covalent interactions and disulfide bonds that are present in the tertiary structure are responsible for stabilizing the multimer that is produced as a result. There are a great number of quaternary structure organizations that could exist. The term multimer refers to complexes that consist of two or more polypeptides, also known as multiple subunits. To be more specific, it would be referred to as a dimer if it consists of two subunits, a trimer if it consists of three subunits, a tetramer if it consists of four subunits, and a pentamer if it consists of five subunits, and so on. There are several instances in which the subunits are connected to one another through symmetry operations, such as a dimer having a twofold axis. The prefix homo- is used to refer to multimers that are composed of identical subunits, whereas the prefix hetero- is used to refer to multimers that are composed of different subunits. For instance, as an example of a heterotetramer, the two alpha chains and the two beta chains of hemoglobin are examples of heterotetramers.

One can use the terms homomer, multimer, or oligomer to refer to a collection of several copies of a certain polypeptide chain.  In the year 2021, Bertolini and colleagues provided findings that suggested that the creation of homomers could be influenced by the interaction between nascent polypeptide chains as they are translated from mRNA by ribosomes that are located in close proximity to one another.  There are hundreds of proteins that have been found to be formed into homomers in human cells after being discovered.  In many cases, the contact of the N-terminal region of polypeptide chains is what kicks off the process of assembly. A review was conducted in 1965 to examine the evidence that multiple gene products form homomers (multimers) in a wide range of animals. This evaluation was based on evidence that was derived via intragenic complementation.

When proteins are discussed, it is common to say that they are composed of several structural units. Folds, domains, and motifs are all examples of these basic units. In spite of the fact that there are approximately 100,000 distinct proteins that are expressed in eukaryotic systems, there are a great deal fewer distinct domains, structural motifs, and folds.

One of the components of the overall structure of a protein is known as a structural domain. This component is self-stabilizing and folds in a manner that is typically independent of the rest of the protein chain. There are a great number of domains that are not exclusive to the protein products of a single gene or gene family, but rather can be found in quite a few different proteins. Because of their significant role in the biological activity of the protein to which they belong, domains are frequently given names and singled out for special attention. For instance, the calcium-binding domain of calmodulin is an example of a domain. Through the use of genetic engineering, domains can be swapped between two proteins in order to create chimeric proteins. This is possible due to the fact that domains exhibit independent stability. It was referred to as a superdomain when it was a conservative combination of numerous domains that exist in various proteins. For example, the protein tyrosine phosphatase domain and the C2 domain pair are examples of domains that can evolve as a single unit.

The term structural and sequence motifs refers to brief regions of the three-dimensional structure of proteins or the sequence of amino acids that were discovered in a wide variety of proteins.

There is the possibility that tertiary protein structures contain several secondary components on the same polypeptide chain. The term supersecondary structure is used to describe a particular combination of elements that come from secondary structures, such as β-α-β units or a motif consisting of a helix-turn-helix. Some of them are also known as structural motifs, which is another name for them.

The term protein fold is used to describe the overall structure of a protein, which may include variations such as a helix bundle, β-barrel, Rossmann fold, or any of the other folds that are included in the Structural Classification of Proteins database. Protein topology is a concept that is connected to this.

Proteins are not fixed entities; rather, they are composed of a diversity of conformational states that cluster together. Transitions between these states often take place on nanoscales, and they have been connected to phenomena that are functionally relevant, such as enzyme catalysis and allosteric signaling. Through the processes of protein dynamics and conformational changes, proteins are able to perform the functions of nanoscale biological machines within cells. These machines typically take the shape of multi-protein complexes. Some examples of motor proteins include myosin, which is responsible for muscle contraction; kinesin, which moves cargo inside cells away from the nucleus along microtubules; and dynein, which moves cargo inside cells towards the nucleus and produces the axonemal beating of motile cilia and flagella. All of these proteins are examples of motor proteins.  [I]n effect, the [motile cilium] is a nanomachine composed of perhaps over 600 proteins in molecular complexes, many of which also function independently as nanomachines...Flexible linkers allow the mobile protein domains connected by them to recruit their binding partners and induce long-range allostery via protein domain dynamics.

Proteins are frequently considered to be relatively stable tertiary structures that undergo conformational changes as a result of being influenced by interactions with other proteins or as a component of enzyme activity. On the other hand, proteins can have variable degrees of stability, and some of the versions that are less stable are proteins that are of an innately disordered nature. There is a relatively 'disordered' state in which these proteins live and function, and they do not possess a stable tertiary structure. As a consequence of this, it is challenging to characterize them using a single defined tertiary structure. For the purpose of providing a more precise and 'dynamic' depiction of the conformational state of proteins that are intrinsically disordered, conformational ensembles have been developed as a method.

A representation of a protein that can be thought of as having a flexible structure is referred to as a protein ensemble file. If you want to create these files, you will need to figure out which of the many different protein conformations that are theoretically possible really exist. The application of computer techniques to the protein data is one method that can be utilized in order to attempt to ascertain the assortment of conformations that are most likely to be present in an ensemble file. Pool and molecular dynamics (MD) approaches are the two primary strategies that can be used to prepare data for the Protein Ensemble Database. The figure illustrates both of these approaches. There are other methods that can be used to prep the data. Within the framework of the pool-based technique, the amino acid sequence of the protein is utilized in order to generate a vast pool of random conformations. Following this, the pool is subjected to additional computational processing, which results in the generation of a set of theoretical parameters for each conformation based on the existing structure. Conformational subsets are chosen from this pool based on the degree to which the average theoretical properties of this protein closely match the experimental data that is already now available. In the alternative molecular dynamics approach, numerous random conformations are taken into consideration at the same time, and each of these conformations is then subjected to experimental data. In this case, the experimental data, which includes things like known distances between atoms, are functioning as limits that can be placed on the conformations. It is only acceptable to accept conformations that are able to stay within the boundaries that have been established by the experimental results. In many cases, this method involves applying a substantial quantity of experimental data to the conformations, which is a task that requires a great deal of computational effort.

The conformational ensembles were created for a number of proteins that are extremely dynamic and partially unfolded. These proteins include Sic1/Cdc4, p15 PAF, MKK7, Beta-synuclein, and P27.

During the process of translation, polypeptides leave the ribosome mostly in the form of a random coil and then fold back into their original configuration. According to Anfinsen's dogma, the ultimate structure of the protein chain is typically thought to be defined by the sequence which the amino acids are arranged in.

Proteins are said to have thermodynamic stability when there is a difference in the amount of free energy that exists between the folded and unfolded states of the protein. This difference in free energy is extremely sensitive to temperature; hence, a change in temperature may result in unfolding or denaturation of the molecule. Protein denaturation can lead to the loss of function as well as the elimination of the protein's original state. In most cases, the free energy required to stabilize soluble globular proteins does not go over fifty kilojoules per mole fraction. When the huge number of hydrogen bonds that occur during the process of stabilizing secondary structures and the hydrophobic interactions that occur during the process of stabilizing the inner core are taken into consideration, the free energy of stabilization is shown to be a minor difference between enormous numbers.

Through the use of X-ray crystallography, approximately ninety percent of the protein structures that are accessible through the Protein Data Bank have been identified. By utilizing this technique, it is possible to ascertain the three-dimensional (3-D) density distribution of electrons within the protein while it is in its crystalline condition. This allows one to infer the three-dimensional coordinates of all the atoms that need to be identified to a particular precision. Nuclear magnetic resonance (NMR) techniques have been used to obtain around seven percent of the protein structures that are now known. Cryo-electron microscopy is able to determine the structures of proteins, regardless of the size of the protein complex. However, the maximum resolution is gradually rising, despite the fact that the resolution is often lower than that of X-ray crystallography or nuclear magnetic resonance (NMR). When it comes to really massive protein complexes, such as those found in viral coat proteins and amyloid fibers, this method continues to be highly successful.

Circular dichroism is a technique that can be utilized to ascertain the general secondary structure composition. Quantification of the conformation of peptides, polypeptides, and proteins can also be accomplished by the utilization of vibrational spectroscopy. There are some structures of flexible peptides and proteins that cannot be investigated using conventional methods; therefore, two-dimensional infrared spectroscopy has become a very useful approach for investigating these structures. It is common practice to gain a more qualitative view of the structure of proteins by the process of proteolysis, which is also helpful in screening for more crystallizable protein samples. There is no requirement for purification when using novel implementations of this technology, such as fast parallel proteolysis (FASTpp), which allows for the investigation of the structured fraction and its relative stability. After the structure of a protein has been identified by experimentation, additional extensive research can be carried out computationally by employing molecular dynamic simulations of that structure.

A database that is modeled around the numerous protein structures that have been determined through experimentation is referred to as a protein structure database. Providing the scientific community with access to experimental data in a manner that is helpful is the primary objective of the majority of protein structure databases. These databases are designed to organize and annotate the protein structures.  Experimental information, such as unit cell dimensions and angles for x-ray crystallography determined structures, are frequently included in the data that is included in protein structure databases. Additionally, 3D coordinates are frequently included in these databases. Although the majority of instances, in this case proteins or specific structure determinations of a protein, also contain sequence information, and some databases even provide means for performing sequence-based queries, the primary characteristic of a structure database is structural information. Sequence databases, on the other hand, are primarily concerned with sequence information and do not include any structural information for the majority of their entries. For many endeavors in computational biology, such as structure-based drug design, protein structure databases are essential. These databases are used for the development of the computational methods that are utilized, as well as for the provision of a large experimental dataset that is utilized by certain methods in order to provide insights on the function of a