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\begin{document}

\title{Biophysical Journal Article Template\\}


\author[ ]{Josh Nicholson}

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\date{}

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\selectlanguage{english}
\begin{abstract}
{{Abstract}}

Insert abstract information here.\\

Insert Received for publication Date and in final form Date\\

Insert Corresponding address and emails\\

Place the Author addresses here\\%
\end{abstract}%



Probing the folding and unfolding processes of proteins as a function of
temperature is a major challenge in biophysics. Here we examine the
effects of temperature spikes that heat and cool proteins within tens of
nanoseconds. Our results show these spikes are capable of causing
irreversible changes sufficient to eliminate protein activity.\\

The folding and unfolding of proteins over milliseconds is commonly
described using conventional chemical kinetics and transition-state
theory (1). On submicrosecond timescales, it has been reported that
proteins are simply too large to undergo the significant structural
changes required for folding, and that the assumptions of conventional
rate kinetics break down (1). ``Speed limits'' for protein folding of
the order of 1\selectlanguage{greek}
{{{{{{μ}{{{s}}}}}}}{μs}}\selectlanguage{english}\textbackslash{}mu\{\textbackslash{}rm s\} have
been reported (2,3). In general, smaller and less complex proteins fold
and unfold more quickly than larger proteins, and most cases of folding
near the ``speed limit'' involve polypeptides
{{{{{{{{\textless{}}}}{100}}}}}{\textless{}100}}\{\textless{}\}100
residues in size.\\

In the case of unfolding, the opportunity exists to substantially
increase the rates by increasing the temperature. ``T-jump'' experiments
have observed unfolding of proteins on microsecond timescales (4,5), and
significant structural change of small proteins in nanoseconds (6).
Molecular dynamics simulations performed at high temperatures
({{{{{{100}{-}{-}{{{{225}}{{{{[?]}}}}}}}}}}{100--225[?]}}100--225\^{}\{\textbackslash{}circ\}C)
have predicted an unfolding ``speed limit'' of
{{{{{{~}}}}}{~}}\textbackslash{}sim0.1 ns (7), but so far no experiments
have been able to show unfolding near this limit.\\

Temperature-jump experiments are restricted to temperatures below the
boiling point of the solution, thus limiting~therate at which unfolding
may occur. Attempts to heat~proteins to higher temperatures have been
made (8), but~these simultaneously induce nonthermal effects (extreme
levels of~electromagnetic fields and microbubbles) likely to affect
proteins. Here, we use a technique that can heat proteins~tocalibrated
temperatures well above
{{{{{{{{{100}}{{{{[?]}}}}}}}}}}{100[?]}}100\^{}\{\textbackslash{}circ\}C
without these~potentially destructive effects. Our measurements achieve
heat/ cool times of 40 ns, with significant cooling within 10~ns.\\

By measuring protein activity we monitor the active site of proteins
without making assumptions about how much secondary structure remains
intact away from the active site. In this article, we define unfolding
to refer to a structural change significant enough to cause loss of
protein activity.\\

All reports of thermally induced unfolding on a nanosecond timescale
have used proteins that unfold reversibly. In this article, nanosecond
temperature spikes are applied to two enzymes, horseradish peroxidase
isozyme C (HRP-C) and catalase, which have previously been shown
toirreversibly unfold when heated (9,10). Temperature spikesbelow
{{{{{{{{{100}}{{{{[?]}}}}}}}}}}{100[?]}}100\^{}\{\textbackslash{}circ\}C do
not affect these enzymes, but at higher temperatures, irreversible
thermal unfolding is induced using temperature spikes lasting only tens
of nanoseconds.\\

We exploit three opportunities for the production of very brief
temperature spikes in solution:\\

\begin{itemize}
\tightlist
\item
  Heat rapidly diffuses away from very thin layers in a heat conductive
  medium. By solving the heat conduction equation, it is readily shown
  that confining a temperature rise to a small structure results in
  extremely fast cooling times.\\
\item
  Metals combine a high thermal conductivity with a large extinction
  coefficient for visible light. For example, a 150 nm gold film on
  glass transmits
  {{{{{{\textless{}}{0.1}{\%}}}}}{\textless{}0.1\%}}\textless{}0.1\textbackslash{}\%
  of incident laser light (Fig. 1 \emph{a}). The film is also thin
  enough to deliver significant heat to the gold surface and the liquid
  medium in contact with it. In this way, ``pure'' conducted heat is
  delivered to macromolecules attached to the gold-liquid interface
  without the complicating effect of large electromagnetic fields, or
  photons with sufficient energy to break covalent bonds.\\
\item
  The boiling of a liquid on a surface requires the formation of active
  vapor nuclei. For a smooth water-metal interface at the temperatures
  and timescales we employ, the rate of vapor generation is negligible
  (11).\\
\end{itemize}

For inserting figures, all the images are in current working folder and
insert the below command for calling figures. We have to insert the
image wherever the first reference in the text.\\\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/biophysj00089789F01-LW/default-figure}
\caption{{\hypertarget{auto-label-caption-439410}{}
Laser heating system used to deliver temperature pulses to proteins
(purple ellipses). Temperature is monitored using the top reflecting
beam (\textbf{red}). (\textbf{b}) Two typical temperature pulses
comparing the calculated transient (\textbf{dashed blue lines}) and the
measured temperature (\textbf{red lines}). Full width at half-maximum of
both modeled spikes is 40 ns.%
}}
\end{center}
\end{figure}

Our apparatus (Fig. 1 \emph{a}) consists of a frequency doubled (532 nm)
Nd:YAG laser with 5 ns pulse length, heating a 135 nm thick gold film
with 15 nm chromium underlayer on a glass substrate. The chromium is
used to increase film adhesion and also increases the absorbance of
laser light.\\

Thermal modeling, previously reported in detail (12),shows that
temperature rises affect an aqueous layer up to 30 nm from the gold
surface (much larger than the protein dimensions). The modeled
temperature profiles are in close agreement with temperature
measurements obtained using a thermoreflectance method. There is a
significant reduction in temperature elevation within 10 ns and a 50\%
reduction in 40 ns (Fig. 1 \emph{b}). The uniformity of surface
temperature is determined by the intensity distribution of the laser
beam, by the uniformity of the gold layer, and by the effect of
interference fringes from the glass substrate. The total nonuniformity
in temperature rise, expressed as a standard deviation of the mean
temperature change, was\selectlanguage{ngerman}
{{{{{{18}{±}{4}{\%}}}}}{\(18 \pm 4\%\)}}18\textbackslash{}pm
4\textbackslash{}\% for experiments with catalase and
{{{{{{25}{±}{5}{\%}}}}}{\(25 \pm 5\%\)}}25\textbackslash{}pm
5\textbackslash{}\% for HRP-C.\\

Catalase is a ubiquitous enzyme that catalyses the hydrolysis of
hydrogen peroxide and whose activity can be simply measured (13). It is
a 250 kDa tetramer, considerably larger than the proteins whose
nanosecond unfolding kinetics have previously been observed (4--6).\\

HRP-C is a 43 kDa enzyme widely used in biotechnology for its ability to
oxidize a variety of substances in a reaction coupled with hydrogen
peroxide. We found that both proteins were enzymatically active when
adsorbed onto gold films and suffered irreversible loss of activity
after ambient heating. Binding of the proteins to the gold surface
reduced protein stability, with the thermal inactivation rate of
catalase on the surface roughly equivalent to that in a solution
{{{{{{{{{10}}{{{{[?]}}}}}}}}}}{\(10^{\circ}\)}}10\^{}\{\textbackslash{}circ\}C
warmer than the surface.\\

The enzymatic activities of catalase and HRP after exposure to thermal
spikes of varying peak temperatures are shown in Fig. 2. The temperature
range over which the loss of function occurs is broadened due to
nonuniformities in the exposure. A 50\% loss of activity was used as the
criterion for assessing the loss of function as this measure was found
to be insensitive to these nonuniformities. For catalase, 50\% loss of
activity occurred after a single temperature pulse reaching\selectlanguage{ngerman}
{{{{{{174}{±}{{{{7}}{{{{[?]}}}}}}}}}}{\(174 \pm 7^{\circ}\)}}\selectlanguage{english}174\textbackslash{}pm
7\^{}\{\textbackslash{}circ\}C (95\% C.I.), or after 100 pulses each
reaching\selectlanguage{english}
{{{{{{139}{+-}{{{{9}}{{{{[?]}}}}}}}}}}{\(139 \pm 9^{\circ}\)}}139\textbackslash{}pm
9\^{}\{\textbackslash{}circ\}C (95\% C.I.). Fig. 2 also presents data
for HRP-C, which show a similar pattern of activity loss, but require
significantly greater temperatures. Loss of activity for HRP-C was
achieved using 100 pulses each producing a temperature of\selectlanguage{english}
{{{{{{{{~}}}{{{{290}}{{{{[?]}}}}}}}}}}{\(\sim 290^{\circ}\)}}\{\textbackslash{}sim\}290\^{}\{\textbackslash{}circ\}C.
Fourier transform infrared measurements of the protein amide I band
before and after heating show that the loss of activity is not due to
removal of protein from the surface.\\\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/biophysj00089789F02-LW/default-figure}
\caption{\selectlanguage{ngerman}{\hypertarget{auto-label-caption-710362}{}
Normalized enzymatic activities for catalase (\textbf{red}) and HRP-C
(\textbf{blue}). Data above \(230\)°C assume a linear
extrapolation of the measured temperature versus pulse energy relation
beyond that point.%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/biophysj00089789F03-LW/default-figure}
\caption{\selectlanguage{ngerman}{\hypertarget{auto-label-caption-232627}{}
Arrhenius plot of the rate of loss of enzymaticactivity of catalase
immobilized at a gold surface (see Fig. 1 \textbf{a}). The data points
in the lower right are obtained by heating in a water bath (error bars
represent mean ±~SE fits to inactivation rate), whereas the upper left
data points used the laser heating apparatus 1 (error bars are 95\%
C.I.). The line of best fit (\textbf{bold}) and 95\% C.I. lines were
determined by fitting the water bath data only.%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

This is example table for four column\\\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
coloumn 1 & column 2 & column 3 & column 4\tabularnewline
\midrule
\endhead
123 & 456 & 789 & --456\tabularnewline
829 & 657 & 987 & 642\tabularnewline
223 & 543 & 9876 & 654\tabularnewline
3212 & 5678 & 9876 & 766\tabularnewline
\bottomrule
\end{longtable}

To elucidate the mechanism involved in catalase inactivation, we have
compared the laser heating of catalase with experiments performed using
ambient heating between
{{{{{{{{{45}}{{{{[?]}}}}}}}}}}{\(45^{\circ}\)}}45\^{}\{\textbackslash{}circ\}C
and
{{{{{{{{{63}}{{{{[?]}}}}}}}}}}{\(63^{\circ}\)}}63\^{}\{\textbackslash{}circ\}C
for catalase attached to identical chromium-gold films. The inactivation
rates observed in this range were fit with a simple Arrhenius equation,
which when extrapolated to higher temperatures predicts inactivation
rates in good agreement with those measured using the laser apparatus
(Fig. 3). The intermediate range of Fig. 3
{{{{{{(}{k}{~}{{{{10}}{{{{[?]}}}}}}{-}{{{{10}}{{{{4}}}}}}{{{{{{s}}}}{{{{-}{1}}}}}}{)}}}}}{\(\left( k \sim 10^{\circ} - 10^{4}s^{- 1} \right)\)}}(k\textbackslash{}sim
10\^{}\{\textbackslash{}circ\}-10\^{}\{4\}\textbackslash{},\{\textbackslash{}rm
s\}\^{}\{-1\}) cannot be measured with either technique used here. This
range would require a prohibitively large number of laser pulses using
the thermal spike technique. Nonetheless, the compatibility of the two
results, using water bath heating and laser-induced temperature spikes,
is strongly suggestive of a similar mechanism for thermally induced
unfolding across a change in inactivation rate of nearly 12 orders of
magnitude.\\

There are strong theoretical reasons to expect that theArrhenius
behavior we observe will not continue indefinitely. Protein unfolding is
likely to be subject to a ``speed limit''. The limit suggested by
molecular dynamics modeling, performed on substantially smaller proteins
than those used here, is of the order of
{{{{{{{{{10}}{{{{10}}}}}}{{{{{{s}}}}{{{{-}{1}}}}}}}}}}{\(10^{10}s^{- 1}\)}}10\^{}\{10\}\textbackslash{},\{\textbackslash{}rm
s\}\^{}\{-1\} (7). We have shown that behavior similar to that predicted
by an Arrhenius equation holds very close to this predicted limit, for
two large proteins (43 and 250 kDa). We have also demonstrated
experimentally that irreversible unfolding of these proteins can occur
due to thermal spikes lasting only tens of nanoseconds in duration, a
timescale significantly faster than the ``speed limit'' associated with
protein folding.\\

The ability to probe protein kinetics at ultrahigh temperatures on a
nanosecond timescale has direct applications to the management of pulsed
exposures to electromagnetic radiation (14), and to the development of
new techniques emerging in laser surgery and medicine (15,16). Our use
of a planar geometry, besides allowing a straightforward measurement of
temperature, has the advantage of enabling surface imaging after
nanosecond temperature spikes. This would provide opportunities to
investigate the kinetic behavior of biological macromolecules such as
motor proteins (17). Moreover, our results provide information about
fundamental protein kinetics at temperatures previously inaccessible to
experiment.\\

\section*{SUPPLEMENTARY MATERIAL}\label{auto-label-section-706221}

An online supplement to this article can be found by visiting BJ Online
at http://www.biophysj.org.

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