Temperature-Induced Changes in Protein Structures Studied by Fourier Transform Infrared Spectroscopy and Global Analysis

Stokkum, Ivo H. M. van; Linsdell, H.; Hadden, Jonathan M.; Haris, Parvez I.; Chapman, D.; Bloemendal, Michael

doi:10.1021/bi00033a024

Cited by 168 publications

(175 citation statements)

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“…Global analysis fitting of the spectra measured as a function of temperature or detergent concentration was performed using a spectral model (35) and constraints on some of the amplitudes. Other fitting of data was performed using a least-squares fitting procedure and programmed in Igor Pro (WaveMetric Inc.).…”

Section: Methodsmentioning

confidence: 99%

Oligomerization of Light-Harvesting I Antenna Peptides of Rhodospirillum rubrum

Pandit

Visschers²,

Stokkum

et al. 2001

Biochemistry

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We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated R BChl 2 subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl--D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperaturemediated transition at low detergent concentration, a set of spectral forms with maxima slightly blueshifted from 873 nm were observed, possibly due to opened rings with one or only a few R BChl 2 units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of -216 kJ mol -1 and an entropy change of -0.59 kJ mol -1 K -1 , at a detergent concentration of 0.8% n-octyl--D-glucopyranoside.The light-harvesting complexes in the membranes of photosynthetic bacteria are responsible for the initial capture of light and transfer of excitation energy to the photosynthetic reaction centers. In purple non-sulfur bacteria, two types of light-harvesting complexes occur: the LH1 1 core antenna surrounding the reaction center and the LH2 peripheral antenna that is connected to the LH1 (1-4). Both types of complexes consist of ringlike oligomers of two types of pigment-protein subunits, the so-called R and polypeptides, that each bind one or two bacteriochlorophyll pigments. For all species, both the R and polypeptide contain one transmembrane R-helical stretch as a highly conserved structural element (1, 5). The structures of LH2 of Rhodopseudomonas. acidophila (6) and LH2 of Rhodospirillum. molischianum (7) have been resolved to high resolution: both complexes were shown to contain an inner ring of R-polypeptides and an outer ring of -polypeptides with a ring of bacteriochlorophylls, absorbing at 850 nm and called B850s, sandwiched between the two rings. Between the -polypeptides, a second ring of bacteriochlorophylls absorbing at 800 nm occurs, and these are called B800s and are positioned parallel to the membrane plane, close to the cytosolic surface. The LH2 ring of ...

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Section: Methodsmentioning

confidence: 99%

Oligomerization of Light-Harvesting I Antenna Peptides of Rhodospirillum rubrum

Pandit

Visschers²,

Stokkum

et al. 2001

Biochemistry

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“…Table 2 shows the band positions and percentage areas of the components of untreated (control) TrwC-N275, TrwC-N275 after transfer to a water bath at 90°C for 10 min and rapid cooling, and TrwC-N275 after being heated to 90°C at a rate of 1°C per min. It is clear that whereas no difference can be detected between the first two samples, the slowly heated protein presents two peaks at 1,683 and 1,618 cm Ϫ1 , which have been found to be characteristic of protein aggregation (1, 2) and which have been attributed to intermolecular hydrogen bonding between extended structures (23). Note that the band at 1,654 cm Ϫ1 , corresponding to the ␣-helix, seems not to be affected by aggregation and that the band at 1,618 cm Ϫ1 arises mainly at the expense of the ␤-sheet structure.…”

Section: Vol 185 2003 Infrared Analysis Of Trwc 4227mentioning

confidence: 98%

A Bacterial TrwC Relaxase Domain Contains a Thermally Stable α-Helical Core

et al. 2003

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The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid Nterminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly ␣/␤ structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the ␣-helix percentage is not altered and ␣-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an ␣-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of ␤-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.Bacterial conjugation is the result of a two-step process, DNA processing and DNA transport. Each step is carried out by a specific set of proteins encoded by the tra genes of a given conjugative plasmid (16). Conjugation begins with the cleavage of the donor supercoiled DNA by a specific relaxase and the formation of a nucleoprotein complex, the relaxosome, which contacts the transport site. A multiprotein DNA transport system effects the transfer process of the cleaved DNA strand to the recipient cell. The relaxase religates the transferred DNA strand upon transfer, and finally, host proteins replicate both single strands in the donor and recipient bacteria to regenerate the double-stranded conjugative plasmid.Relaxases are classified into five families according to the DNA sequences around the nic sites and their amino acid sequences (9). The F family relaxase TrwC, from the IncW plasmid R388, is a dimeric protein of 996 amino acids in which the N-terminal domain has a DNA relaxase activity and the C-terminal domain is a DNA helicase (18). In addition to relaxase and helicase activities, the TrwC protein promotes site-specific recombination between oriT sequences in vivo. In addition, it can cleave a supercoiled plasmid DNA containing oriT in vitro in the absence of accessory proteins (17).Infrared (IR) spectroscopy has become a widely used tool in the study of protein structure. In principle, a structure as large as a protein would give rise to an enormous number of overlapping vibrational modes, obscuring the information that could be obtained in practice, but because o...

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“…Room temperature ATR-FTIR spectra of higher antibody concentrations are consistent with the high intramolecular b-sheet content of these proteins with peak positions of the zero order spectra at 1635 cm À1 (spectra not illustrated). 18 The second derivative spectra of 10 mg/mL MAb 1 and 2 solutions exhibit two peaks between 1620 and 1640 cm À1 . In the spectra of 10 mg/mL MAb 1 solutions, the peaks are at 1638 and 1628 cm À1 (Fig.…”

Section: Ftir Spectroscopymentioning

confidence: 99%