Ultrafast Biophysical Studies of Biomolecules at the NIH

随着美国国立卫生研究院开始放松对返回实验室的要求，我们合作的压力跳跃核磁共振研究的步伐加快了。目前有三个国产的压力跳跃系统可供使用。在大流行期间，用于操作高场布鲁克核磁共振磁体的控制台进行了升级，这需要对压力跳变装置控制系统进行额外修改。例如，压力跳变装置的控制器响应正常的TTL信号，但升级后的核磁共振控制台产生低压TTL信号（LV-TTL），这是不兼容的。为了适应这种变化，我们制作了一个供电电路，将来自NMR控制台的LV-TTL信号转换为正常TTL信号，并恢复了压力跳变装置的功能。Baber博士是一名支持LCP核磁共振工作的科学家，他接受过培训，可以接管这些系统的日常监督，并被证明是这次合作中非常有能力和受欢迎的成员。我们共同改进和标准化了用于快速切换样品池压力的高速气动阀控制器的设计。此外，他还组装了额外的传输线，因此我们每次安装时都有备用的传输线。现在，当实验过程中发生泄漏时，更换传输线并恢复数据采集相对简单，干扰最小。压力跳变装置实现了阿梅洛素寡聚化的环力研究，该研究结果最近发表在：“实验NOE，化学位移和丙氨酸异构化数据提供了阿梅洛素寡聚化的详细见解”，Sai Chaitanya Chiliveri, Yang Shen, James L. Baber, Jinfa Ying, Vatsala Sagar, Graeme Wistow， Philip Anfinrud和Ad Bax；化学学报，2014,32,1806318074 （10.1021/jacs.3c05710）。（注：本文已在网上发表，但尚未分配Pubmed ID。）

As the NIH began loosening requirements for returning to the lab, the pace of our collaborative pressure-jump NMR studies picked up. Three home-built pressure-jump systems are currently operable. During the pandemic, the consoles used to operate the high-field Bruker NMR magnets were upgraded, which required additional modifications to the pressure-jump apparatus control systems. For example, the controller for the pressure-jump apparatus responds to normal TTL signals, but the upgraded NMR console generates low voltage TTL signals (LV-TTL), which are incompatible. To accommodate this change, we fabricated a powered circuit that converts LV-TTL signals from the NMR console to normal TTL signals and restored the functionality of the pressure-jump apparatus. Dr. Baber, a staff scientist who supports NMR efforts in LCP has been trained to take over day-to-day oversight of these systems and has proven a very capable and welcome addition to this collaboration. Together, we have improved and standardized the design of the high-speed pneumatic valve controllers used to rapidly switch the sample cell pressure. Moreover, he assembled additional transfer lines so we always have a spare ready to go at each installation. Now, when a leak develops during an experiment, it is relatively straightforward to swap out the transfer line and resume data acquisition with minimal disruption. The pressure-jump apparatus enabled a tour-de-force study of amelotin oligomerization, which was recently published: "Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization", Sai Chaitanya Chiliveri, Yang Shen, James L. Baber, Jinfa Ying, Vatsala Sagar, Graeme Wistow, Philip Anfinrud, and Ad Bax; Journal of the American Chemical Society, 145, 32, 1806318074 (10.1021/jacs.3c05710). (Note: this manuscript has been published online, but has not yet been assigned a Pubmed ID.) A very important parameter when pursuing time-resolved studies is the sample temperature. For example, life prospers at temperatures as low as -1.8 C for arctic fish to over 116 C for bacteria found in the vicinity of geothermal vents. Since rates of reaction are strongly temperature dependent, biomolecules have evolved to perform their intended function at the temperature of their environment. To investigate biomolecule structural dynamics over a large temperature range, we developed a novel temperature controller capable of precisely setting the temperature of a sample capillary over a range of temperatures spanning -16 - 120 C. By etching the 300 micron ID capillary with HF and filtering the sample solution before loading, we avoid nucleation of water ice and can super cool our samples to -16 C without freezing. By pressuring the sample to 3 atm, we extend our temperature range up to 120 C without boiling. The temperature controller operates a pair of thermoelectric coolers (TECs) that employ the Peltier effect to pump heat to/from an aluminum nozzle that surrounds the capillary and controls its temperature. To avoid condensation on the capillary at low temperatures, we flow a dry gas through the nozzle and achieve laminar flow around the capillary. The temperature stability achieved in the capillary can be as good as a few mK. One of our modes of data acquisition involves what we call a Tramp, in which the temperature repeatedly ramps between low and high temperature settings at a slew rate of 1 C per second, with results from several successive Tramps averaged to improve the S/N of the data. However, we found that operating the TECs at a high temperature leads to propagation of micro cracks in the semiconductor pillars that separate the hot and cold surfaces, which in turn degrades their performance. To maintain peak performance, we have had to replace the TECs frequently, which is akin to replacing an engine in a car. We hypothesized that damage to the TEC may be a consequence of strain induced when the temperature difference between the hot and cold surfaces of the TEC is large, especially while at high temperature. As a proof of principle, we performed a large number of Tramps between 35 and 120 C with the TEC heat sink heated to 75 C, which limited the temperature difference across the TEC to a maximum of 45 C. No degradation of the TEC performance was observed after executing hundreds of Tramp sequences. Hence, we are in the process of upgrading our temperature control system with two water sources, one hot ( 75 C) and one cold ( 4 C), and plan to employ an electronically controlled valve to direct the appropriate temperature water into the TEC heat sink. By switching the heat sink water source during the Tramp, we limit the temperature difference across the TEC and should thereby maintain its performance, hopefully indefinitely. We aim to finalize the development of our temperature controller in the coming year and publish the design parameters that enable us to achieve unprecedented temperature control over a wide range of temperatures without TEC degradation. A new post-doc in our group, Dr. Eli Worth (arrived in March 2023), is helping develop a compact, general-purpose, dual-beam, time-resolved absorption spectrometer capable of characterizing with high sensitivity structural dynamics in photoactive biomolecules over a broad range of temperatures and time scales. This spectrometer employs a 12-bit resolution digital oscilloscope that will track signals from high-gain 150 MHz bandwidth photodiodes. This bandwidth is sufficient to achieve time resolution down to about 2 ns. Preliminary studies indicate that our S/N ratio is approaching the shot-noise limit. Moreover, the memory depth of the oscilloscope is sufficient to acquire data over a very large dynamic range of times after a single laser shot. We are currently using a fixed-frequency diode laser as the probe, but are awaiting delivery of a picosecond pulsed super continuum laser source that will allow scanning the probe wavelength over most of the visible spectrum and well into the near IR. The sample environment consists of a capillary with a 100 micron square cross section through which sample will be introduced via a syringe pump. The capillary will be positioned in the nozzle of our home-built temperature controller to provide temperature control from -16 to 120 Celsius. An off-axis parabolic mirror is used to focus pump pulses from up to three different pump lasers onto the sample. One of the pump sources is an Opolette HE 355 LD optical parametric oscillator, which is capable of generating laser pulses of 5 ns duration over a very broad range of wavelengths spanning from 410 to 2400 nm. A second source consists of a 527 nm CW laser that is gated with an acousto-optic modulator (AOM) and can switch on/off in about 35 ns. The aim is to use the short pulse laser to initially photolyze a sample, and the AOM-switched laser will be used to maintain the photolyzed state. This home-built instrument should prove quite useful to characterize the time scales over which biomolecules undergo structural transitions and will help identify sample conditions appropriate for subsequent studies with more advanced techniques, such as time-resolved x-ray scattering and diffraction.