A protein's amino acid sequence determines its three-dimensional structure ( conformation). In turn, a protein's structure determines the function of that protein. Preface 1 An Introduction to protein structure and function A brief and very selective historical perspective The biological diversity of proteins Proteins and the. Your article () from "Proteins: Structure, Function, and Bioinformatics" is available for download After printing the PDF file, please read the page proofs.
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CHAPTER 4. Proteins: Structure, Function, Folding. – Structure and properties of the peptide bond. – Structural hierarchy in proteins. – Structure and function of. Primary Structure of Proteins. The amino acid sequence or primary structure of a purified protein can be determined. Polypeptide sequences can be obtained . Protein Structure. Page 2. Amino Acids. Amino acids are the building blocks of proteins. All AA's . The structure, function and general properties of a protein are.
The final shape of the protein complex is once again stabilized by various interactions, including hydrogen-bonding, disulfide-bridges and salt bridges. The four levels of protein structure are shown in Figure 2. Due to the nature of the weak interactions controlling the three-dimensional structure, proteins are very sensitive molecules.
The term native state is used to describe the protein in its most stable natural conformation in situ. This native state can be disrupted by a number of external stress factors including temperature, pH, removal of water, presence of hydrophobic surfaces, presence of metal ions and high shear. The loss of secondary, tertiary or quaternary structure due to exposure to a stress factor is called denaturation.
Denaturation results in unfolding of the protein into a random or misfolded shape. A denatured protein can have quite a different activity profile than the protein in its native form, usually losing biological function.
In addition to becoming denatured, proteins can also form aggregates under certain stress conditions. Aggregates are often produced during the manufacturing process and are typically undesirable, largely due to the possibility of them causing adverse immune responses when administered.
In addition to these physical forms of protein degradation, it is also important to be aware of the possible pathways of protein chemical degradation. These include oxidation, deamidation, peptide-bond hydrolysis, disulfide-bond reshuffling and cross-linking. The methods used in the processing and the formulation of proteins, including any lyophilization step, must be carefully examined to prevent degradation and to increase the stability of the protein biopharmaceutical both in storage and during drug delivery.
The complexities of protein structure make the elucidation of a complete protein structure extremely difficult even with the most advanced analytical equipment. An amino acid analyzer can be used to determine which amino acids are present and the molar ratios of each.
The sequence of the protein can then be analyzed by means of peptide mapping and the use of Edman degradation or mass spectroscopy. This process is routine for peptides and small proteins, but becomes more complex for large multimeric proteins. Peptide mapping generally entails treatment of the protein with different protease enzymes in order to chop up the sequence into smaller peptides at specific cleavage sites.
Two commonly used enzymes are trypsin and chymotrypsin. Mass spectroscopy has become an invaluable tool for the analysis of enzyme digested proteins, by means of peptide fingerprinting methods and database searching.
Edman degradation involves the cleavage, separation and identification of one amino acid at a time from a short peptide, starting from the N-terminus. One method used to characterize the secondary structure of a protein is circular dichroism spectroscopy CD.
These spectra can be used to approximate the fraction of the entire protein made up of each type of structure. A more complete, high-resolution analysis of the three-dimensional structure of a protein is carried out using X-ray crystallography or nuclear magnetic resonance NMR analysis.
To determine the three-dimensional structure of a protein by X-ray diffraction, a large, well-ordered single crystal is required. X-ray diffraction allows measurement of the short distances between atoms and yields a three-dimensional electron density map, which can be used to build a model of the protein structure.
The use of NMR to determine the three-dimensional structure of a protein has some advantages over X-ray diffraction in that it can be carried out in solution and thus the protein is free of the constraints of the crystal lattice.
Many different techniques can be used to determine the stability of a protein. For the analysis of unfolding of a protein, spectroscopic methods such as fluorescence, UV, infrared and CD can be used. Thermodynamic methods such as differential scanning calorimetry DSC can be useful in determining the effect of temperature on protein stability.
HPLC is also an invaluable means of analyzing the purity of a protein. Other analytical methods such as SDS-PAGE, iso-electric focusing and capillary electrophoresis can also be used to determine protein stability, and a suitable bioassay should be used to determine the potency of a protein biopharmaceutical.
The variety of methods for determining protein stability again emphasizes the complexity of the nature of protein structure and the importance of maintaining that structure for a successful biopharmaceutical product.
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Volume 8 PDF Version Increasingly, drug developers are looking to large molecules and particularly proteins as a therapeutic option.
Secondary Structure Stretches or strands of proteins or peptides have distinct characteristic local structural conformations or secondary structure , dependent on hydrogen bonding. Tertiary Structure The overall three-dimensional shape of an entire protein molecule is the tertiary structure.
Quaternary Structure Many proteins are made up of multiple polypeptide chains, often referred to as protein subunits. Protein Stability Due to the nature of the weak interactions controlling the three-dimensional structure, proteins are very sensitive molecules.
Protein Structure Analysis The complexities of protein structure make the elucidation of a complete protein structure extremely difficult even with the most advanced analytical equipment. Protein Structure Stability Analysis Many different techniques can be used to determine the stability of a protein.
References 1. Murphy 2. Shirley Particle Sciences is a leading integrated provider of formulation and analytic services and both standard and nanotechnology approaches to drug development and delivery.
When combined in various sequences, this array of functional groups accounts for the broad spectrum of protein function. For instance, the chemical reactivity associated with these groups is essential to the function of enzymes, the proteins that catalyze specific chemical reactions in biological systems see Chapters 8— Proteins can interact with one another and with other biological macromolecules to form complex assemblies. The proteins within these assemblies can act synergistically to generate capabilities not afforded by the individual component proteins Figure 3.
These assemblies include macro-molecular machines that carry out the accurate replication of DNA , the transmission of signals within cells, and many other essential processes. Some proteins are quite rigid, whereas others display limited flexibility.
Rigid units can function as structural elements in the cytoskeleton the internal scaffolding within cells or in connective tissue. Parts of proteins with limited flexibility may act as hinges, springs, and levers that are crucial to protein function, to the assembly of proteins with one another and with other molecules into complex units, and to the transmission of information within and between cells Figure 3.
Crystals of human insulin. Insulin is a protein hormone, crucial for maintaining blood sugar at appropriate levels. Below Chains of amino acids in a specific sequence the primary structure define a protein like insulin. These chains fold into well-defined more Structure Dictates Function.
The structure of the protein allows large segments of DNA to be copied without the replication machinery dissociating from the more A Complex Protein Assembly. An electron micrograph of insect flight tissue in cross section shows a hexagonal array of two kinds of protein filaments.
Michael Reedy. Flexibility and Function. Upon binding iron, the protein lactoferrin undergoes conformational changes that allow other molecules to distinguish between the iron-free and the iron-bound forms. By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed. Turn recording back on.
National Center for Biotechnology Information , U. New York: W H Freeman; W H Freeman ;