Janne Savolainen

Rational Design of an Active Avidin Monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 → 2 interface was mutated to lysine and asparagine 54 in the 1 → 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K d ∼10−15 m of the wild-type tetramer to K d ∼10−7 m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.

Pump-probe and pump-deplete-probe spectroscopies on carotenoids with N=9–15 conjugated bonds

A series of all-trans-carotenoids with N=9, 13, and 15 conjugated bonds has been studied by pump-probe and pump-deplete-probe spectroscopies to obtain a systematic analysis of the energy flow between the different electronic states. The ultrafast dynamics in the carotenoids are initialized by excitation to the S2 state and subsequently manipulated by an additional depletion pulse in the near-IR spectral range. The changes in the dynamics after depletion of the excited state population allowed differentiation of the excited state absorption into two components, a major one corresponding to the well known S1 state and the small contribution on the red wing of the S0-S2 absorption band originating from the hot ground state. We found no evidence for an additional electronically excited state, usually called S*. Instead, a deactivation mechanism that includes the hot ground state supports the observed results nicely in the framework of a simple three state model (S2, S1, and S0).

Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

In order to turn the subunit association and biotin binding of avidin into pH‐sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1–3 interface, and in combination with a histidine conversion in the 1–2 interface (Trp110). The single replacements Met96His and Val115His in the 1–3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH‐dependent. The histidine replacement in the 1–2 interface affected the biotin‐binding properties of the mutants, in particular reversibility of binding and protein–ligand complex formation were pH‐sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin–biotin technology.

Controlling the efficiency of an artificial light-harvesting complex.

Adaptive femtosecond pulse shaping in an evolutionary learning loop is applied to a bioinspired dyad molecule that closely mimics the early-time photophysics of the light-harvesting complex 2 (LH2) photosynthetic antenna complex. Control over the branching ratio between the two competing pathways for energy flow, internal conversion (IC) and energy transfer (ET), is realized. We show that by pulse shaping it is possible to increase independently the relative yield of both channels, ET and IC. The optimization results are analyzed by using Fourier analysis, which gives direct insight to the mechanism featuring quantum interference of a low-frequency mode. The results from the closed-loop experiments are repeatable and robust and demonstrate the power of coherent control experiments as a spectroscopic tool (i.e., quantum-control spectroscopy) capable of revealing functionally relevant molecular properties that are hidden from conventional techniques.

Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment.

Chicken avidin and bacterial streptavidin are proteins used in a wide variety of applications in the life sciences due to their strong affinity for biotin. A new and promising use for them is in medical pretargeting cancer treatments. However, their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in these applications. To search for potentially beneficial new candidates, we screened egg white from four different poultry species for avidin. Avidin proteins, isolated from the duck, goose, ostrich and turkey, showed a similar tetrameric structure, similar glycosylation and stability against both temperature and proteolytic activity of proteinase K as chicken avidin. Biotin-binding properties of these avidins, measured using IAsys optical biosensor, were similar to those found in avidin from the chicken. Three of these novel avidins, however, showed different immunological cross-reactivities when compared with chicken avidin. The patient sera responses to duck, goose and ostrich avidins were also lower than those observed for chicken and turkey avidins. Our findings suggest that the use of these proteins offers advantages over chicken avidin and bacterial streptavidin in pretargeting applications.

Two-dimensional-Raman-terahertz spectroscopy of water: Theory

We discuss the hybrid 2D-Raman-THz spectroscopy of liquid water. This two-dimensional spectroscopy is designed to directly work in the low-frequency range of the intermolecular degrees of freedom. The information content of 2D-Raman-THz spectroscopy is similar to 2D-Raman or 2D-THz spectroscopy, but its experimental implementation should be easier. That is, 2D-Raman-THz spectroscopy is a 3rd-order nonlinear spectroscopy and as such completely avoids cascading of consecutive 3rd-order signals, which turned out to be a major difficulty in 5th-order 2D-Raman spectroscopy. On the other hand, it does not require any intense THz pump-pulse, the lack of which limits 2D-THz spectroscopy to the study of semiconductor quantum wells as the currently available pulse energies are too low for molecular systems. In close analogy to 2D-Raman spectroscopy, the 2D-Raman-THz response of liquid water is simulated from an all-atom molecular dynamics simulation, and the expected spectral features are discussed.

Ultrafast Energy Transfer Dynamics of a Bioinspired Dyad Molecule

A caroteno-purpurin dyad molecule was studied by steady-state and pump-probe spectroscopies to resolve the excited-state deactivation dynamics of the different energy levels as well as the connecting energy flow pathways and corresponding rate constants. The data were analyzed with a two-step multi-parameter global fitting procedure that makes use of an evolutionary algorithm. We found that following ultrafast excitation of the donor (carotenoid) chromophore to its S2 state, the energy flows via two channels: energy transfer (70%) and internal conversion (30%) with time constants of 54 and 110 fs, respectively. Additionally, some of the initial excitation is found to populate the hot ground state, revealing another limitation to the functional efficiency. At later times, a back transfer occurs from the purpurin to the carotenoid triplet state in nanosecond timescales. Details of the energy flow within the dyad as well as species associated spectra are disentangled for all excited-state and ground-state species for the first time. We also observe oscillations with the most pronounced peak on the Fourier transform spectrum having a frequency of 530 cm(-1). The dyad mimics the dynamics of the natural light-harvesting complex LH2 from Rhodopseudomonas acidophila and is hence a good model system to be used in studies aimed to further explain previous work in which the branching ratio between the competing pathways of energy loss and energy transfer could be manipulated by adaptive femtosecond pulse shaping.

Evolution strategies for laser pulse compression

This study describes first steps taken to bring evolutionaryoptimization technology from computer simulations to real world experimentationin physics laboratories. The approach taken considers a wellunderstood Laser Pulse Compression problem accessible both to simulationand laboratory experimentation as a test function for variants ofEvolution Strategies. The main focus lies on coping with the unavoidablenoise present in laboratory experimentation. Results from simulations arecompared to previous studies and to laboratory experiments.

Robust orthogonal parameterization of evolution strategy for adaptive laser pulse shaping.

Many spectroscopic applications of femtosecond laser pulses require properly-shaped spectral phase profiles. The optimal phase profile can be programmed on the pulse by adaptive pulse shaping. A promising optimization algorithm for such adaptive experiments is evolution strategy (ES). Here, we report a four fold increase in the rate of convergence and ten percent increase in the final yield of the optimization, compared to the direct parameterization approach, by using a new version of ES in combination with Legendre polynomials and frequency-resolved detection. Such a fast learning rate is of paramount importance in spectroscopy for reducing the artifacts of laser drift, optical degradation, and precipitation.

Evolution Strategies for Laser Pulse Compression

This study describes first steps taken to bring evolutionaryoptimization technology from computer simulations to real world experimentationin physics laboratories. The approach taken considers a wellunderstood Laser Pulse Compression problem accessible both to simulationand laboratory experimentation as a test function for variants ofEvolution Strategies. The main focus lies on coping with the unavoidablenoise present in laboratory experimentation. Results from simulations arecompared to previous studies and to laboratory experiments.