Protein Structure and Conformational Studies

The Far UV region of a protein circular dichroism (CD) spectrum shows spectral features related to the structure of the backbone of the protein, and is directly relevant to the secondary structural elements that make up the protein structure. The spectral signatures can be used to predict the secondary structure makeup of a particular proteins structure. This is probably the most well known known application of CD spectroscopy, and there has been a number of analytical packages produced to aid in predicting secondary structure using circular dichroism data.

Far-UV CD spectrum thermal denaturation of lysozyme

The above figure illustrates clearly the change in CD signature as a function of temperature. Comparison of known CD signatures with the data in this experiment suggests that the initial CD signature is a combination of α-helical and β-sheet structure which, on heating, loses much of it’s α-helical content to an unfolded state, whilst retaining some β-sheet character.

Secondary structure prediction is only part of the power of circular dichroism spectroscopy. CD spectra in the near UV regions of the CD spectrum are caused by side chain residues of the phenylalanine, tryptophan and tyrosine residues. When these residues are in highly structured environments (buried in a structured protein) they show distinct structured CD spectra, conversely when in unstructured environments (in bulk solution after unfolding of a protein for instance) they have very little structured CD spectral features. Changes in the CD spectra of the near UV show changes in the structure around these residues because of changes in the structure of the protein.

Changes in the circular dichroism spectra of bio-molecules represent changes in their secondary and tertiary structures. When this is coupled with the facts that (i) spectra can be recorded in minutes and (ii) single wavelength kinetics can be recorded from milliseconds onwards, it can be seen that CD is a particularly powerful tool to follow dynamic changes in protein structure. For instance, changes induced by changing temperature, pH, ligands, or denaturants are all commonly studied.

Another powerful application of circular dichroism is to simply compare two proteins, and determine if they have a similar structure. This can be used simply to ascertain if a newly purified protein is correctly folded, determine if a mutant protein has folded correctly in comparison to the wild-type, or for the analysis of biopharmaceutical products to determine that the biopharmaceutical is still in a correctly folded active conformation.

Relevant Circular Dichroism References

Listed below are 5 selected recent references of studies of various protein structural using APL circular dichroism systems. A complete searchable database with all references can be accessed by logging into the APL members area.

Authors	Title	Year	Keywords	Journal/Proceedings
Sylvie Campagna, Nathalie Saint, Gérard Molle, and André Aumelas	Structure and Mechanism of Action of the Antimicrobial Peptide Piscidin [Abstract]   [URL]	2007	Antibacterial Peptide, Structure and Mechanism, circular dichroism, NMR, secondary structure	BIOCHEMISTRY-USA, 2007, Vol 46, Iss 7, pp 1771-1778
Abstract: Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high α-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an α-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.
Castelletto, V., Krysmann, M., Kelarakis, A. & Jauregi, P.	Complex Formation of Bovine Serum Albumin with a Poly(ethylene glycol) Lipid Conjugate [Abstract]   [URL]	2007	Self-assembly, bovine serum albumin, formulation, circular dichroism, small-angle x-ray scattering	BIOMACROMOLECULES, 2007, Vol 8, Iss 7, pp 2244-2249
Abstract: In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.
Sachin Kale, Palaniappa Arjunan, William Furey, and Frank Jordan	A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component [Abstract]   [URL]	2007	crystallographic studies, Escherichia coli, kinetic, spectroscopic, and crystallographic studies.	J. Biol. Chem., Vol. 282, Issue 38, 28106-28116, September 21, 2007
Abstract: Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.
Anna L. Mallam and Sophie E. Jackson	The Dimerization of an α/β-Knotted Protein Is Essential for Structure and Function [Abstract]   [URL]	2007	Structure and function, α/β-Knotted proteins, biological self-assembly, polypeptide .	Structure, Volume 15, Issue 1, January 2007, Pages 111-122.
Abstract: α/β-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the α/β-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein.
Denis B. D. O'SULLIVAN, Christopher E. JONES, Salama R. ABDELRAHEIM, Andrew R. THOMPSETT, Marcus W. BRAZIER, Harold TOMS, David R. BROWN and John H. VILES	NMR characterization of the pH 4 β-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility [Abstract]   [URL]	2007	Prion diseases, misfolding, prion protein, α-helical isoform , β-sheet-rich oligomer.	Biochem. J. (2007) 401 (533–540)
Abstract: Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely α-helical isoform to a β-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an a-helical protein into one that contains a high proportion of β-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. 15N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23–231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional 15N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23–118. 15N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112–231), which shows only 16 15N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.