**2.2 Experimental determination of protein structure**

The basic prerequisite for understanding the function of a protein is the knowledge of the protein 3D structure. The experimental methods used in the study of tertiary structure include:


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*Role of Force Fields in Protein Function Prediction DOI: http://dx.doi.org/10.5772/intechopen.93901*

**2.3 Protein structure prediction**

achieved by three different ways:

iii.Hierarchical methods.

*2.3.1 Approaches based on energy minimization*

cryo-EM is split randomly into half and the two averages (or 3D reconstructions) over rings (or shells, respectively) are compared, with increasing radius in Fourier space using an appropriate amount of reproducibility [13]. The protein sample in EM and cryo-EM does not require crystallization, saving a lot of time and effort, which is a major advantage over protein x-ray crystallography. Nevertheless, for membrane proteins, electron crystallography is used which require two dimensional (2D) crystals of the sample protein. Another advantage of cryo-EM is that it requires very less amount of sample materials. However, one of the limitations of cryo-EM is that it has to compromise with the resolution comparative to resolution obtained from

The field of structural biology is mostly dominated by experimental methods which are expensive and laborious in nature. However, since the last few decades, the application of computational techniques in structural biology has been widely used, with significant improvements in these techniques since last 10–20 years. This has helped to achieve substantial developments in protein structure prediction methods. *In-silico* protein structure prediction enables the prediction of 3D structures for proteins with known sequences and unknown structures. Prediction of the tertiary structure also helps in understanding the folding and unfolding of proteins. Also, protein engineering may help in incorporation of new functions in proteins thus facilitating drug design and discovery [15]. Protein structure prediction can be

i.Computer simulation-based on empirical energy minimization

ii.Knowledge based-approaches using information derived from known sequences of experimentally determined protein 3-D structures

The energy minimization method is also known as the *ab-initio* (*de* novo) method for protein structure prediction and is based on the theory that the native structure of protein is always at thermodynamic equilibrium with minimum energy, which is calculated using basic laws of physics and chemistry (**Figure 1**). Energy minimizationbased methods always attempt to detect the global minima in free energy surface of the protein molecule as it is thought that global minima correspond to the native conformation. This method is not very helpful to design protein sequence length of more than 150 amino acid residues. However, it can be used to design small stable peptides that can bind to any specific therapeutic targets [16]. Two types of energy minimization methods are broadly used in *de novo* structure prediction approach, namely static and dynamical minimization methods. Some of the major FF used for energy minimizations are GROMOS, AMBER, CHARMM and ECEPP [17, 18]. One of the *ab-initio* protein structure prediction software packages is ROSETTA. This software package is based on the postulation that local interactions lead the conformation of short segments while global interactions establish the 3D protein structure [19]. The advantage of *ab-initio* approach is that it is based on physicochemical principles, however, these principles are hampered by the vast number of degrees of freedom which are needed to be looked after and also the performance of energy functions are

x-ray crystallography and NMR spectroscopy [14].

*Homology Molecular Modeling - Perspectives and Applications*

**2.2 Experimental determination of protein structure**

tertiary structure include:

travels to the detector through a flight tube. The ions with lighter mass will reach the detector faster due to Newton's second law (F = ma) and hence, will be detected first. After the spectrum is recorded, it is further analyzed and compared against a database of sequenced proteins. A detailed sequence of protein fragments can be determined by repeating the process with different enzymes for cleavage. As a result, the fragments become much smaller with the

fragments overlapping each other establishing the order of the protein.

The basic prerequisite for understanding the function of a protein is the knowledge of the protein 3D structure. The experimental methods used in the study of

i.*Protein X-ray crystallography*: X-ray crystallography is presently the most sought-after technique for determination of biological macromolecule structures. In this method, the determination of protein structure is achieved by crystallization of the purified protein at high concentration and exposing the crystals to an X-ray beam. The resultant diffraction patterns, obtained from the diffraction spots, are then processed to get knowledge about the symmetry of the packaging of the crystal and the size of the repeating units forming the crystal. A map of the electron density is then calculated using the "structure features", which are determined from the intensities of the diffraction spots. The quality of the electron density map can be improved using various methods. This is done to get a definitive idea to build the molecular structure using the amino acid sequence. Finally, the structure that is obtained is further refined to fit the map more accurately and to assume a conformation which is thermodynamically more favorable. Protein crystallography is known to provide highly accurate protein structures by giving atomic resolution. However, this method is not always straightforward and may take

a lot of time to complete, which is around 3–5 years [11].

ii.*Nuclear magnetic resonance (NMR spectroscopy)*: Another useful technique to determine the protein structure is the NMR spectroscopy. It is a primary quantitative method which allows concentration determination of proteins in an aqueous environment that may resemble its actual physiological state more closely. In principle, the NMR spectroscopy is dependent on the electromagnetic radiation and the sample protein interaction. It is used to observe the local magnetic fields prevailing around the protein atomic nuclei. The NMR signal is obtained when sensitive radio receivers detect the excitation of the material nuclei with radio waves into the nuclear magnetic resonance. Thus, it provides access to the electronic structure of the sample protein. The major advantage of NMR over X-ray crystallography is that the protein in NMR spectroscopy can be examined in their native-like physiological state. However, NMR is not suitable for proteins with more than 150 amino acid and needs the protein under study to be stable in room temperature for a long

time of data acquisition, which is a drawback of this technique [12].

iii.*Electron microscopy (especially Cryo-electron microscopy)*: Electron microscopy (EM) and cryo-electron microscopy (cryo-EM) are used to study objects that are comparatively larger in size such as cellular organelles or large macromolecular complexes with higher resolution. EM and cryo-EM use a method known as single-particle reconstruction. In principle, the data set in EM and

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cryo-EM is split randomly into half and the two averages (or 3D reconstructions) over rings (or shells, respectively) are compared, with increasing radius in Fourier space using an appropriate amount of reproducibility [13]. The protein sample in EM and cryo-EM does not require crystallization, saving a lot of time and effort, which is a major advantage over protein x-ray crystallography. Nevertheless, for membrane proteins, electron crystallography is used which require two dimensional (2D) crystals of the sample protein. Another advantage of cryo-EM is that it requires very less amount of sample materials. However, one of the limitations of cryo-EM is that it has to compromise with the resolution comparative to resolution obtained from x-ray crystallography and NMR spectroscopy [14].
