Protein Structure and Function - Biochemistry - NCBI Bookshelf
Does protein function depend on the linear sequence of amino acids? Its Three -Dimensional Structure; Summary; Appendix: Acid-Base Concepts; Problems. Structure-function relationships in proteins and DNA: definition of the problem . Biological processes in any living organism are based on selective. Understanding how cells work requires understanding how proteins function. attempts at crystallization, a common problem for many membrane proteins.
Selenium is highly reactive, so this amino acid must be handled with care: Structurally, selenocysteine looks just like cysteine, but with a —SeH group in place of the —SH group 4 4. Peptide bonds Each protein in your cells consists of one or more polypeptide chains. Each of these polypeptide chains is made up of amino acids, linked together in a specific order. The chemical properties and order of the amino acids are key in determining the structure and function of the polypeptide, and the protein it's part of.
But how are amino acids actually linked together in chains?Hemoglobin and myoglobin
The amino acids of a polypeptide are attached to their neighbors by covalent bonds known as a peptide bonds. Each bond forms in a dehydration synthesis condensation reaction.
During protein synthesisthe carboxyl group of the amino acid at the end of the growing polypeptide chain chain reacts with the amino group of an incoming amino acid, releasing a molecule of water. The resulting bond between amino acids is a peptide bond Peptide bond formation between two amino acids.
The question of what a protein does inside a living cell is not a simple one to answer. Imagine isolating an uncharacterized protein and discovering that its structure and amino acid sequence suggest that it acts as a protein kinase. Simply knowing that the protein can add a phosphate group to serine residues, for example, does not reveal how it functions in a living organism.
Additional information is required to understand the context in which the biochemical activity is used. Where is this kinase located in the cell and what are its protein targets? In which tissues is it active? Which pathways does it influence? What role does it have in the growth or development of the organism? In this sectionwe discuss the methods currently used to characterize protein structure and function.
We begin with an examination of the techniques used to determine the three-dimensional structure of purified proteins. We then discuss methods that are used to predict how a protein functions, based on its homology to other known proteins and its location inside the cell. Finally, because most proteins act in concert with other proteins, we present techniques for detecting protein-protein interactions. But these approaches only begin to define how a protein might work inside a cell.
In the last section of this chapter, we discuss how genetic approaches are used to dissect and analyze the biological processes in which a given protein functions.
Introduction to proteins and amino acids
It is presently not possible, however, to deduce reliably the three-dimensional folded structure of a protein from its amino acid sequence unless its amino acid sequence is very similar to that of a protein whose three-dimensional structure is already known.
The main technique that has been used to discover the three-dimensional structure of molecules, including proteins, at atomic resolution is x-ray crystallography. X-rays, like light, are a form of electromagnetic radiation, but they have a much shorter wavelength, typically around 0. If a narrow parallel beam of x-rays is directed at a sample of a pure proteinmost of the x-rays pass straight through it.
A small fraction, however, is scattered by the atoms in the sample. If the sample is a well-ordered crystal, the scattered waves reinforce one another at certain points and appear as diffraction spots when the x-rays are recorded by a suitable detector Figure Figure X-ray crystallography.
A A narrow parallel beam of x-rays is directed at a well-ordered crystal B. Shown here is a protein crystal of ribulose bisphosphate carboxylase, an enzyme with a central role in CO2 fixation during photosynthesis. Some of the more The position and intensity of each spot in the x-ray diffraction pattern contain information about the locations of the atoms in the crystal that gave rise to it. Deducing the three-dimensional structure of a large molecule from the diffraction pattern of its crystal is a complex task and was not achieved for a protein molecule until But in recent years x-ray diffraction analysis has become increasingly automated, and now the slowest step is likely to be the generation of suitable protein crystals.
This requires large amounts of very pure protein and often involves years of trial and error, searching for the proper crystallization conditions. There are still many proteins, especially membrane proteins, that have so far resisted all attempts to crystallize them. Analysis of the resulting diffraction pattern produces a complex three-dimensional electron -density map. Interpreting this map—translating its contours into a three-dimensional structure—is a complicated procedure that requires knowledge of the amino acid sequence of the protein.
Largely by trial and error, the sequence and the electron-density map are correlated by computer to give the best possible fit.
The reliability of the final atomic model depends on the resolution of the original crystallographic data: A complete atomic model is often too complex to appreciate directly, but simplified versions that show a protein 's essential structural features can be readily derived from it see Panelpp. The three-dimensional structures of about 10, different proteins have now been determined by x-ray crystallography or by NMR spectroscopy see below —enough to begin to see families of common structures emerging.
These structures or protein folds often seem to be more conserved in evolution than are the amino acid sequences that form them see Figure X-ray crystallographic techniques can also be applied to the study of macromolecular complexes. In a recent triumph, the method was used to solve the structure of the ribosomea large and complex cellular machine made of several RNAs and more than 50 proteins see Figure The determination required the use of a synchrotron, a radiation source that generates x-rays with the intensity needed to analyze the crystals of such large macromolecular complexes.
This technique is now also increasingly applied to the study of small proteins or protein domains. Unlike x-ray crystallography, NMR does not depend on having a crystalline sample; it simply requires a small volume of concentrated protein solution that is placed in a strong magnetic field.
Certain atomic nuclei, and in particular those of hydrogen, have a magnetic moment or spin: The spin aligns along the strong magnetic field, but it can be changed to a misaligned, excited state in response to applied radiofrequency RF pulses of electromagnetic radiation. When the excited hydrogen nuclei return to their aligned state, they emit RF radiation, which can be measured and displayed as a spectrum. The nature of the emitted radiation depends on the environment of each hydrogen nucleusand if one nucleus is excited, it influences the absorption and emission of radiation by other nuclei that lie close to it.
It is consequently possible, by an ingenious elaboration of the basic NMR technique known as two-dimensional NMR, to distinguish the signals from hydrogen nuclei in different amino acid residues and to identify and measure the small shifts in these signals that occur when these hydrogen nuclei lie close enough together to interact: In this way NMR can give information about the distances between the parts of the protein molecule.
By combining this information with a knowledge of the amino acid sequence, it is possible in principle to compute the three-dimensional structure of the protein Figure Figure NMR spectroscopy.
A An example of the data from an NMR machine.
Rediscovering Biology - Online Textbook: Unit 2 Proteins & Proteomics
This two-dimensional NMR spectrum is derived from the C-terminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein more For technical reasons the structure of small proteins of about 20, daltons or less can readily be determined by NMR spectroscopy.
Resolution is lost as the size of a macromolecule increases. But recent technical advances have now pushed the limit to aboutdaltons, thereby making the majority of proteins accessible for structural analysis by NMR. The NMR method is especially useful when a protein of interest has resisted attempts at crystallization, a common problem for many membrane proteins.
Because NMR studies are performed in solution, this method also offers a convenient means of monitoring changes in protein structure, for example during protein folding or when a substrate binds to the protein.
NMR is also used widely to investigate molecules other than proteins and is valuable, for example, as a method to determine the three-dimensional structures of RNA molecules and the complex carbohydrate side chains of glycoproteins.
Some landmarks in the development of x-ray crystallography and NMR are listed in Table Sequence Similarity Can Provide Clues About Protein Function Thanks to the proliferation of protein and nucleic acid sequences that are catalogued in genome databases, the function of a gene —and its encoded protein—can often be predicted by simply comparing its sequence with those of previously characterized genes. Because amino acid sequence determines protein structure and structure dictates biochemical function, proteins that share a similar amino acid sequence usually perform similar biochemical functions, even when they are found in distantly related organisms.
At present, determining what a newly discovered protein does therefore usually begins with a search for previously identified proteins that are similar in their amino acid sequences.
Searching a collection of known sequences for homologous genes or proteins is typically done over the World-Wide Web, and it simply involves selecting a database and entering the desired sequence. A sequence alignment program—the most popular are BLAST and FASTA—scans the database for similar sequences by sliding the submitted sequence along the archived sequences until a cluster of residues falls into full or partial alignment Figure The results of even a complex search—which can be performed on either a nucleotide or an amino acid sequence—are returned within minutes.
Such comparisons can be used to predict the functions of individual proteins, families of proteins, or even the entire protein complement of a newly sequenced organism. Sequence databases can be searched to find similar amino acid or nucleic acid sequences. In the end, however, the predictions that emerge from sequence analysis are often only a tool to direct further experimental investigations.
Search term Chapter 3Protein Structure and Function Proteins are the most versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. They function as catalysts, they transport and store other molecules such as oxygen, they provide mechanical support and immune protection, they generate movement, they transmit nerve impulses, and they control growth and differentiation.
Indeed, much of this text will focus on understanding what proteins do and how they perform these functions. Several key properties enable proteins to participate in such a wide range of functions.
Proteins are linear polymers built of monomer units called amino acids. The construction of a vast array of macromolecules from a limited number of monomer building blocks is a recurring theme in biochemistry. Does protein function depend on the linear sequence of amino acids?