Hadden, Jonathan Mark; (1995) Protein structure and conformational changes studied by Fourier transform infrared spectroscopy. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Fourier transform infrared spectroscopy (FTIR) has been used to study a range of different proteins. These include:- i) Human serum transferrin, human lactoferrin and rabbit serum transferrin. A study has been made of the structural changes that accompany iron binding and release from these related proteins. Structural variations within this group of proteins have been shown. Thermal denaturation studies, using differential scanning calorimetric measurements, have been related to FTIR spectral changes and indicate that the crystal structure of iron-free human lactoferrin may not reflect the structure of this protein in solution. ii) Human placental transferrin receptor. This protein has been examined at extracellular and endosomal pH. Both the intact protein in detergent and its water soluble major extracellular fragment have been investigated. iii) The bacterial adhesive protein streptococcal antigen I/II. The solution structure and thermal stability of the native protein, the recombinant full-length protein and four recombinant fragments of this protein have been examined using both Fourier transform infrared spectroscopy and circular dichroism spectroscopy. iv) Albumin, IgG, Ribonuclease, Fibrinogen, A study of the thermal stability of these and other proteins has been undertaken to compare their denaturation properties in H2O and 2H2O solutions. v) Lysozyme, Ribonuclease These and several other proteins have been examined using Fourier transform infrared microscopy in order to compare the infrared spectra of these proteins, both in solution and also in the form of single crystals. Many of the proteins show similar spectra, whether recorded in solution or from a single crystal. In some cases, as with single crystals oi Endothia parasitica pepsin, Mucor pucillus pepsin and serum amyloid P component, the spectra differ from those recorded in solution. These differences may indicate that a rearrangement of turns structures occurs upon crystallisation. The major novel findings of this work are: i) There are small but significant structural differences between human serum transferrin, rabbit serum transferrin and human lactoferrin. The crystal structure of human lactoferrin may not represent the structure of this protein in solution. The apparent conflict between CD, FTIR and X-ray crystallographic estimates of secondary structural content of the transferrins can be explained by the fact that CD estimates are based on dihedral angles and not hydrogen bonding patterns. ii) Intact transferrin receptor precipitates out of solution at endosomal pH while the extracellular fragment remains soluble but undergoes a conformational change. This results in minor change in the secondary structural content of the protein and a reduction of thermal stability by approximately 15°C. iii) The recombinant polypeptides produced to study the structure of streptococcal antigen I/II have been shown to fold into defined secondary structures. Furthermore the structures of these fragments have been used to predict a possible structure for the native protein. iv) Quantitative analysis of thermally induced changes in the FTIR spectra of proteins in H2O solution may not offer any significant advantage to analyses performed in 2H2O due to problems associated with the different molar absorption coefficients of the separate secondary structures in H2O solution. v) Infrared spectroscopy/microscopy can be successfully be applied to the study of protein structure in both the solution and crystalline form.

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