Instrumentation sequence structures

Because of its structural simplicity and instrumental role in protein function, the coiled coil is one of the most investigated protein folding motifs. Native coiled-coil sequences and their mutants have been synthesized and studied. Numerous model coiled-coil peptides have been designed de novo [22]. Although many theoretical questions remain unanswered, much has been learned about the sequence-structure relationship. It is even possible to design and engineer new coiled-coil sequences and structures that have never existed before [23]. 

A reevaluation of the original sequence data established that natural bradykinm was indeed the nonapeptide shown Here the synthesis of a peptide did more than confirm structure synthesis was instrumental m determining structure... 

Microstructure studies, by contrast, offer both a means to evaluate the reactivity ratios and also to test the model. The capability to investigate this type of structural detail was virtually nonexistent until the advent of modern instrumentation and even now is limited to sequences of modest length. 

To determine the structure of a protein or peptide, we need to answer three questions What amino acids are present How much of each is present in what sequence do the amino acids occur in the peptide chain The answers to the first two questions are provided by an automated instrument called an amino acid analyzer. 

This chapter has reviewed the application of ROA to studies of unfolded proteins, an area of much current interest central to fundamental protein science and also to practical problems in areas as diverse as medicine and food science. Because the many discrete structure-sensitive bands present in protein ROA spectra, the technique provides a fresh perspective on the structure and behavior of unfolded proteins, and of unfolded sequences in proteins such as A-gliadin and prions which contain distinct structured and unstructured domains. It also provides new insight into the complexity of order in molten globule and reduced protein states, and of the more mobile sequences in fully folded proteins such as /1-lactoglobulin. With the promise of commercial ROA instruments becoming available in the near future, ROA should find many applications in protein science. Since many gene sequences code for natively unfolded proteins in addition to those coding for proteins with well-defined tertiary folds, both of which are equally accessible to ROA studies, ROA should find wide application in structural proteomics. 

I The most important properties of a protein are deter-f mined by the sequence of amino acids in the polypeptide chain. This sequence is called the primary structure of the protein. We know the sequences for thousands of peptides and proteins, largely through the use of methods developed in Fred Sanger s laboratory and first used to determine the sequence of the peptide hormone insulin in 1953. Knowledge of the amino acid sequence is extremely useful in a number of ways (1) it permits comparisons between normal and mutant proteins (see chapter 5) (2) it permits comparisons between comparable proteins in different species and thereby has been instrumental in positioning different organisms on the evolutionary tree (see fig. 1.24) (3) finally and most important, it is a vital piece of information for determining the three-dimensional structure of the protein. 

Even if we restrict our design to a small number of sites in the protein, the combinatorial possibilities quickly approach astronomical dimensions. If we consider mutations at 10 sites and a subset of 10 amino acids, we have 1010 possible variants. Although experimental approaches are under development that can actually search large subsets of protein sequence space, it is not at all a small feat to identify those variants that give rise to a stable structure and at the same time come close to the desired features. Therefore, computational approaches that, with some reliability, are able to pick those variants having a stable structure are desirable instruments in the protein engineer s toolbox. 

The purpose of an analytical study is to obtain information about some object or substance. The substance could be a solid, a liquid, a gas, or a biological material. The information to be obtained can be varied. It could be the chemical or physical composition, structural or surface properties, or a sequence of proteins in genetic material. Despite the sophisticated arsenal of analytical techniques available, it is not possible to find every bit of information of even a very small number of samples. For the most part, the state of current instrumentation has not evolved to the point where we can take an instrument to an object and get all the necessary information. Although there is much interest in such noninvasive devices, most analysis is still done by taking a part (or portion) of the object under study (referred to as the sample) and analyzing it in the laboratory (or at the site). Some common steps involved in the process are shown in Figure 1.1. 

Once the sample preparation is complete, the analysis is carried out by an instrument of choice. A variety of instruments are used for different types of analysis, depending on the information to be acquired for example, chromatography for organic analysis, atomic spectroscopy for metal analysis, capillary electrophoresis for DNA sequencing, and electron microscopy for small structures. Common analytical instrumentation and the sample preparation associated with them are listed in Table 1.1. The sample preparation depends on the analytical techniques to be employed and their capabilities. For instance, only a few microliters can be injected into a gas chromatograph. So in the example of the analysis of pesticides in fish liver, the ultimate product is a solution of a few microliters that can be injected into a gas chromatograph. Sampling, sample preservation, and sample preparation are... 

---